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International Journal of

Molecular Sciences Review

Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions Oussama Ahrazem 1 , Lourdes Gómez-Gómez 1 , María J. Rodrigo 2 , Javier Avalos 3 and María Carmen Limón 3, * 1

2

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Instituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain; [email protected] (O.A.); [email protected] (L.G.-G.) Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Departamento de Ciencia de los Alimentos, Calle Catedrático Agustín Escardino 7, 46980 Paterna, Spain; [email protected] Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain; [email protected] Correspondence: [email protected]; Tel.: +34-954-555947

Academic Editor: Vladimír Kˇren Received: 5 September 2016; Accepted: 8 October 2016; Published: 26 October 2016

Abstract: Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms. Keywords: algae; apocarotenoids; bacteria; carotenoid cleavage dioxygenase; fungi; plants

1. Introduction Carotenoids are a large group of terpenoid fat-soluble pigments widely distributed in nature. They are abundantly present in plants, where they are masked by the green color of chlorophyll, but they are responsible for the yellow, orange, or reddish colors of many fruits and flowers. They provide color also to many microorganisms and to some animals, including birds, fishes, and crustaceans. Chemically, the carotenoids are C40 polyene compounds with a chain of conjugated double bonds, which creates a chromophore that absorbs light in the UV and blue range of the spectrum. Carotenoids are produced by algae and plants as well as by many fungi and bacteria [1–3]. Animals are mostly unable to synthesize carotenoids, but an outstanding exception was found in certain aphids, explained by recent carotenoid biosynthetic gene horizontal transfer from fungi [4,5]. Carotenoids exert important biological functions in most living organisms, usually related with their photoprotective and light-absorbing properties. Their functions are especially relevant in plants, algae, cyanobacteria, and anoxygenic prototrophic bacteria [4,6,7], where they are indispensable in photosynthesis because of their light harvesting and protecting roles [8]. Further, carotenoids act as precursors of a vast group of bioactive compounds, the apocarotenoids, with very diverse biological functions [9]. Int. J. Mol. Sci. 2016, 17, 1781; doi:10.3390/ijms17111781

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The electron-rich polyene chain of carotenoids makes them susceptible to oxidative breakdown, leading to the generation of unspecific apocarotenoid products by random cleavage, carried out by carotenoid-unrelated enzymes such as lipoxygenases or peroxidases. However, apocarotenoids are usually the result of a biologically active process, resulting from the action of specific carotenoid cleavage dioxygenases (CCDs), frequently referred to as oxygenases (CCOs), a family of non-heme iron-type enzymes that cleave double bonds in the conjugated carbon chain of carotenoids [10]. Although the first carotenoid cleaving activity was reported in animal tissues [ 11], the first CCD was identified in the plant Arabidopsis thaliana [12], initiating the discovery of a large series of CCD enzymes in many other species. CCDs typically catalyze the cleavage of non-aromatic double bonds by dioxygen to form aldehyde or ketone products. Some CCDs act specifically on apocarotenoid substrates, and these enzymes are known as apocarotenoid cleavage oxygenases (ACOs). In addition to carotenogenic organisms, represented by plants, algae, fungi, and bacteria, CCDs are also widespread in animals, using them to cleave carotenoids acquired through the diet. This review covers the different CCD families identified hitherto in microorganisms and in photosynthetic species. In the microbial sections, the name CCDs will be generically used to include all types of oxygenases, and the nomenclature ACO will be reserved for the apocarotenoid specific oxygenases. In the plant section, we will refer to the CCD1, 2, 4, 7, and 8 enzyme subfamilies. The members of the nine-cis-epoxycarotenoid dioxygenases (NCED) subfamily responsible for the specific cleavage of 9-cis-epoxycarotenoids and involved in the production of abscisic acid (ABA) are not included in this review since it has been the subject of numerous studies and their activities and functions are well known [12–18]. 2. Bacterial Carotenoid Oxygenases Biological roles of CCDs in bacteria are not well established. In cyanobacteria, apocarotenoids act as photoprotective and accessory pigments in thylakoid membranes. Cyanobacteria are known also to be responsible for undesired flavors in drinking water and fish from aquiculture. They produce different odor compounds such as fermentation products and apocarotenoids, the former of which is also found in microbially produced dairy food. The apocarotenoids include derivatives of β-ionone, which generally exhibit pleasant odors found in many flowers and are widely used by industry. Apocarotenoids are produced from carotenoids by CCDs, the first of which was described in Microcystis PPC 7806 [19]. A very different function is found, however, in some archaea and eubacteria, where these enzymes are essential for the biosynthesis of retinal, the chromophore for rhodopsins, or similar pumps [20–22]. In fungi, a similar function has been also described (see fungal section). 2.1. Structural Studies The first crystal structure of a CCD was determined for an apocarotenoid cleavage oxygenase (ACO) from Synechocystis sp. PCC 6803 [23]. The spatial organization resembles a propeller with seven blades, conserved in all described CCDs and, in fact, a structural signature for all of them. Five blades (I to V) are made of four antiparallel β strands, and two blades (VI and VII) consist of 5 strands (Figure 1) [24]. The active center is located on the top of the enzyme, close to the propeller axis. CCDs contain 2+ a Fe ion as a cofactor that is indispensable for the cleavage activity. Its putative role is to activate oxygen involved in the enzymatic reaction. The Fe2+ is coordinated by four His residues, which are conserved in the CCD family. There is a second coordination center formed by three Glu residues interacting through hydrogen bonds to three of the His residues. The requirement for these amino acids has been demonstrated via mutagenesis [25–27]. Another characteristic of CCDs is a large tunnel perpendicular to the propeller axis that enters the protein, passes through the active center, and exits the protein parallel to the propeller axis. The access to the tunnel is important for the entrance of the substrate and is located in a large hydrophobic patch that allows for the localization of the enzyme in the cell membrane. This long

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patch that allows for the localization of the enzyme in the cell membrane. This long tunnel consists tunnel consists of residues hydrophobic and a few aromatic residues (Tyr, Trp, His), of hydrophobic (Phe, residues Val, Leu)(Phe, and aVal, fewLeu) aromatic residues (Tyr, Trp, His), forming “van forming “van der Waals” forces allowing a correct orientation of the substrate [24]. The hydrophobic der Waals” forces allowing a correct orientation of the substrate [24]. The hydrophobic and aromatic andresidues aromaticplay residues play an important in isomerase activity, demonstrated through mutagenesis an important role in role isomerase activity, demonstrated through mutagenesis experiments [24]. experiments [24].

Figure 1. Cont.

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Figure 1. Tridimensional models of 12 carotenoid-cleavage dioxygenases from all the subfamilies Figure 1. Tridimensional models of 12 carotenoid-cleavage dioxygenases from all the subfamilies included in this review. The VP14 (PBD: 2biwA) structure from maize has been used as a template. included in this review. The VP14 (PBD: 2biwA) structure from maize has been used as a template. (A) Side view of CCDs with β-strands shown in yellow, α-helices in magenta, and loops in grey; (A) Side view of CCDs with β-strands shown in yellow, α-helices in magenta, and loops in grey; (B) Top view rotated 90° towards the viewer from (A); (C) Lateral and top views of CCD2, CCD8, and (B) Top view rotated 90◦ towards the viewer from (A); (C) Lateral and top views of CCD2, CCD8, ACO showing Fe2+ ion in green and histidines in blue. Accession numbers are: VP14: O24592.2, ACOX, and ACO showing Fe2+ ion in green and histidines in blue. Accession numbers are: VP14: O24592.2, P74334; AtCCD1, O65572; AtCCD7, AEC10494.1; AtCCD8, Q8VY26; AtCCD4: O49675; Cao-2, ACOX, P74334; AtCCD1, O65572; AtCCD7, AEC10494.1; AtCCD8, Q8VY26; AtCCD4: O49675; XP001727958.1; CarS, ADU04395.1; CarX, CAH70723.1; CsCCD2L, ALM23547.1; CcCCD4b1, Cao-2, XP001727958.1; CarS, ADU04395.1; CarX, CAH70723.1; CsCCD2L, ALM23547.1; CcCCD4b1, XP006424046; AcaA, 77754. XP006424046; AcaA, 77754.

The propeller-forming β-strands are conserved between ACO (Synechocystis) and NOV2 The propeller-forming β-strands are conserved betweendifferences ACO (Synechocystis) (Novosphingobium aromaticivorans). However, there are structural in the entranceand loopNOV2 and (Novosphingobium aromaticivorans). However, there are structural differences in the entrance loop the dome of both enzymes. The differences in the residues of these structural domains are seemingly and the dome of both enzymes. The differences in the residues of these structural domains involved in their different substrate specificities. In fact, the Synechocystis model suggests differencesare seemingly involved in their different substrate specificities. InIn fact, thebesides Synechocystis modeltunnel, suggests in the substrate requirement compared with the NOV model. ACO, the substrate there areintwo tunnels made mainly by hydrophobic residues that the active site to differences theother substrate requirement compared with the NOV model. In connect ACO, besides the substrate a hydrophilic Thetunnels reaction products areby directed to the residues cytosol through the mouth of thesite tunnel, there are mouth. two other made mainly hydrophobic that connect the active tunnel. to aexit hydrophilic mouth. The reaction products are directed to the cytosol through the mouth of the exit tunnel. 2.2. Substrate Specificity 2.2. Substrate StudiesSpecificity on bacterial CCDs usually focused the attention on the purification of different enzymes and the determination their specificity through their incubation with diverse carotenoid substrates. Studies on bacterialofCCDs usually focused the attention on the purification of different enzymes Frequently, the enzymes exhibit a high specificity, cleaving at a certain the polyenesubstrates. chain, and the determination of their specificity through their incubation withposition diverseofcarotenoid while others are less specific. The available information is summarized below. Frequently, the enzymes exhibit a high specificity, cleaving at a certain position of the polyene chain,

while others are less specific. The available information is summarized below. 2.2.1. Apocarotenoid Cleavage Oxygenases (ACO) 2.2.1. Apocarotenoid Oxygenases (ACO) double bonds of apo-β-carotenals. The bestACO enzymesCleavage cleave exclusively at C15–C15′ known are cleave Diox1 exclusively from Synechocystis sp. PCC 6803bonds and NosACO, from Nostoc The sp. PCC 7120 0 double ACO ACOs enzymes at C15–C15 of apo-β-carotenals. best-known (Table 1). Diox1 cleaves at the C15–C15′ double bond of certain all-trans-apocarotenoids, such as ACOs are Diox1 from Synechocystis sp. PCC 6803 and NosACO, from Nostoc sp. PCC 7120 (Table 1). apo-β-carotenals, apo-β-carotenols, apo-8′-lycopenal, and apo-lycopenol [28,29]. The enzyme is Diox1 cleaves at the C15–C150 double bond of certain all-trans-apocarotenoids, such as apo-β-carotenals, rather unspecific, since it accepts apo-β-carotenals, apo-β-carotenols, and their 3-hydroxy derivatives, apo-β-carotenols, apo-80 -lycopenal, and apo-lycopenol [28,29]. The enzyme is rather unspecific, since it with lengths ranging from C25 (12′-apo) to C35 (4′-apo), to generate a C20 retinal (or 3-hydroxy-retinal) accepts apo-β-carotenals, apo-β-carotenols, and their 3-hydroxy derivatives, with lengths ranging

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Int. J. Mol. Sci. 2016, 17, 1781 5 of as 38 and a dialdehyde product (between C5 to C15) (Table 1). However, larger substrates, such Int. J. Mol. Sci.(C 2016, 5 of as 38 and a dialdehyde product (between β-carotene 40), 17, are1781 not cleaved [29]. C5 to C15) (Table 1). However, larger substrates, such Int. J.J.Mol. Sci.Sci. 2016, 17, 5 ofsp. 38 Int. 17, 5 of 38 and aMol. dialdehyde product (between to Cof15)the (Table However, larger substrates,Nostoc such as β-carotene (C 402016, ),also are1781 not1781 cleaved [29]. NosACO, named NSC2 [30], C is5 one three1). CCDs of the cyanobacterium andJ. Mol. a dialdehyde product (between 5 one to Cof15different )the (Table However, larger substrates, such as Int. Sci.(C 2016, 17, 5from ofsp. 38 β-carotene 40), are1781 not cleaved [29]. NosACO, also named NSC2 [30], C isexhibit three1). CCDs of preferences, the cyanobacterium Nostoc (strain PCC 7120). The three enzymes substrate as expected and a dialdehyde product (between 5 one to Cof15NosACO )different (Table However, largerorsubstrates, such as β-carotene 40), are1781 not cleaved [29]. Int. J. Mol. Sci.(C 2016, 17, 5from ofsp. 38 NosACO, also named NSC2 [30], is the three1). CCDs of the cyanobacterium Nostoc (strain PCC 7120). The three enzymes exhibit substrate preferences, as expected cooperative functions among them [31].C In vitro, cleaves monocyclic acyclic carotenoids β-carotene (C 40 ), are not cleaved [29]. and a dialdehyde product (between C 5 to C 15 ) (Table 1). However, larger substrates, such as 0 0 NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. Int. J. Mol. Sci. 2016, 17, 1781 5 of 38 and a dialdehyde (strain 7120). The enzymes exhibit different substrate preferences, as expected cooperative functions among [31]. In vitro, NosACO cleaves monocyclic or NosACO acyclic carotenoids at C15–C15′ double bonds generate retinal. In vivo, ableaching activity was from also from CPCC -apo) tothree Cto (4 -apo), to generate C20 retinal (or of 3-hydroxy-retinal) 25 (12 35them NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. β-carotene (C 40), are not cleaved [29]. and a dialdehyde product (between C 5exhibit to C 15NosACO ) (Table 1). However, larger substrates, such as (strain PCC 7120). The three enzymes different substrate preferences, as expected from Int. J. Mol. Sci. 2016, 17, 1781 5 of 38 cooperative functions among them [31]. In vitro, cleaves monocyclic or acyclic carotenoids at C15–C15′ double bonds to generate retinal. In vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1). product (between Ccleaved toNSC2 Cenzymes )[30], (Table 1).C However, larger substrates, such as β-carotene (C40 ), are not 5three 15 (strain PCC 7120). The exhibit substrate preferences, as expected and a dialdehyde (between C 5retinal. to )In (Table 1). However, larger substrates, such as NosACO, also named isIn one of15different the three CCDs of the cyanobacterium Nostoc sp. β-carotene (C 40), areproduct not [29]. cooperative functions among them [31]. vitro, NosACO cleaves monocyclic or acyclic carotenoids at C15–C15′ double bonds to generate vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).from cleaved [29]. cooperative functions among them [31]. In vitro, cleaves monocyclic or acyclic carotenoids β-carotene (C 40), areproduct not cleaved [29]. (strain PCC 7120). The three enzymes exhibit different substrate preferences, asand expected NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. and a dialdehyde (between C 5retinal. to C 15NosACO )In (Table 1). However, larger substrates, such as at C15–C15′ double bonds to generate vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).from Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 NosAco at C15–C15′ double bonds tothem generate retinal. In vivo, bleaching activity of NosACO was also NosACO, named NSC2 [30], is one of NosACO theone three CCDs of the cyanobacterium Nostoc sp. cooperative functions among [31]. In vitro, cleaves monocyclic or[32,33] acyclic carotenoids (strain PCC 7120). The three enzymes exhibit different substrate preferences, asand expected β-carotene (C 40 ),also arealso not cleaved [29]. observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial (Table 1).from NosACO, named NSC2 [30], is of the three CCDs of the cyanobacterium Nostoc sp. Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 NosAco showing products to different substrates. observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).from (strain PCC 7120). The three enzymes exhibit different substrate preferences, asand expected at C15–C15′ double bonds tothem generate retinal. In vivo, bleaching activity of NosACO was also cooperative among [31]. vitro, cleaves monocyclic or acyclic carotenoids NosACO, also named NSC2 [30], isIn one of NosACO the three CCDs of the cyanobacterium Nostoc sp. as expected from Table 1.functions Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 NosAco showing products to different substrates. (strain PCC 7120). The three enzymes exhibit different substrate preferences, Diox1 NosAco Table Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases NosAco cooperative among [31]. In vitro, NosACO cleaves monocyclic or acyclic carotenoids observed on1.functions β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).from at C15–C15′ double bonds tothem generate retinal. In vivo, bleaching activity Diox1 of NosACO was also showing products to different substrates. (strain PCC 7120). The three enzymes exhibit different substrate preferences, asand expected Substrates Synechocystis sp. PCC 6803 cleaves Nostoc sp. PCC 7120 or acyclic carotenoids at Diox1 NosAco cooperative functions among them [31]. In vitro, NosACO monocyclic Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases and(Table NosAco showing products to different substrates. Int. J.on Mol. Sci.Substrates 2016, 17, 1781 5 of 37 at C15–C15′ double bonds tothem generate In vivo, bleaching activity Diox1 of NosACO was observed β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] 1). also cooperative functions among [31]. Inretinal. vitro, NosACO cleaves monocyclic or acyclic carotenoids Synechocystis sp. PCC 6803 NostocNosAco sp. PCC 7120 0 double β-apo-4′-carotenal C15–C15 bonds toof generate retinal. InDiox1 vivo, bleaching activity of NosACO was also observed showing products to different substrates. Table 1. β-carotene, Enzymatic activity bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco Substrates observed on zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1). at C15–C15′ double bonds to generate retinal. In vivo, bleaching activity of NosACO was also Diox1 Synechocystis sp. PCC 6803 NostocNosAco sp. PCC 7120 β-apo-4′-carotenal Substrates showing products to different substrates. Table Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1). such as andon1.aβ-carotene, dialdehyde product (between C5 to C 15)diapocarotenedial (Table larger substrates, observed zeaxanthin, torulene, lycopene, and [32,33] (Table 1). Retinal Not detected Synechocystis sp. PCC 6803 1). However, Nostoc sp. PCC 7120 Diox1 NosAco β-apo-4′-carotenal

Substrates showing Table 1. products Enzymatic activity bacterial [29]. apo-carotenoid cleavage Diox1 and NosAco Retinal Not detected β-carotene (C40to),different are notofsubstrates. cleaved β-apo-4′-carotenal Synechocystis sp. PCC oxygenases 6803 Nostoc sp. PCC 7120 Diox1 NosAco Substrates showing products to activity differentof substrates. Table 1. Enzymatic bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco Retinal Not detected β-apo-4′-carotenal Synechocystis sp. the PCCthree 6803 cleavage Nostoc sp. 7120 Diox1 and also named [30], is apo-carotenoid one of CCDs ofNosAco thePCC cyanobacterium Nostoc sp. TableNosACO, 1.β-apo-8′-carotenal Enzymatic activityNSC2 of bacterial oxygenases NosAco Diox1 Substrates Retinal Not detected showing products to different substrates. β-apo-4′-carotenal Synechocystis sp.different PCC 6803 substrate NostocNosAco sp. PCC 7120 as expected from Diox1 (strain PCC 7120). The three enzymes exhibit preferences, showing products to different substrates. β-apo-8′-carotenal Substrates Retinal Not detected β-apo-4′-carotenal sp. PCC 6803 Nostoc sp. PCC 7120 Diox1 NosAco Retinal Retinal cooperative functions among them Synechocystis [31]. In vitro, monocyclic β-apo-8′-carotenal RetinalNosACO cleavesNot detected or acyclic carotenoids Substrates β-apo-4′-carotenal Synechocystis sp. PCC 6803 NostocRetinal sp. PCC 7120 Retinal β-apo-8′-carotenal at C15–C15′ double bonds to generate retinal. bleaching of Nostoc NosACO was7120 also Substrates Diox1 Synechocystis sp. PCC 6803Not activity NosAco sp. PCC RetinalIn vivo, detected β-apo-4′-carotenal Retinal Retinal β-apo-8′-carotenal 0 -carotenal zeaxanthin, torulene, observed on β-carotene, lycopene, and diapocarotenedial [32,33] (Table 1). Retinal Not detected β-apo-4 β-apo-10′-carotenal Retinal Retinal β-apo-8′-carotenal Retinal NotRetinal detected Retinal Not detected β-apo-10′-carotenal Retinal β-apo-8′-carotenal Retinal Retinal Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco β-apo-10′-carotenal Retinal Retinal β-apo-8′-carotenal Retinal Retinal β-apo-10′-carotenal 0 -carotenal showing products to different substrates. Retinal Retinal β-apo-8 β-apo-8′-carotenal Retinal Retinal β-apo-10′-carotenal Retinal Retinal Retinal Diox1 Retinal β-apo-12′-carotenal Retinal Retinal NosAco β-apo-10′-carotenal Substrates Retinal Retinal β-apo-12′-carotenal Retinal Retinal Synechocystis sp. PCC 6803 Nostoc sp. PCC 7120 β-apo-10′-carotenal Retinal Not detected 0 -carotenal β-apo-12′-carotenal Retinal Retinal β-apo-10 β-apo-4′-carotenal β-apo-10′-carotenal Retinal Not detected β-apo-12′-carotenal NotRetinal detected Retinal Retinal Retinal Retinal β-apo-10′-carotenal Retinal Not detected β-apo-12′-carotenal Retinal Retinal (3R)-3-OH-β-apo-8′-carotenal Retinal Not detected β-apo-12′-carotenal Retinal 0β-apo-8′-carotenal (3R)-3-OH-β-apo-8′-carotenal Retinal NotRetinal detected β-apo-12 -carotenal β-apo-12′-carotenal Retinal Retinal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8′-carotenal Retinal Not detected Retinal Not detected β-apo-12′-carotenal (3R)-3-OH-β-apo-8′-carotenal 3-OH-retinal 3R-3-OH-retinal Retinal Not detected β-apo-10′-carotenal β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8′-carotenal Retinal Not detected 0 Retinal Retinal (3R)-3-OH-β-apo-8 -carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-12′-carotenal (3R)-3-OH-β-apo-8′-carotenal Retinal Not detected (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8′-carotenal β-apo-12′-carotenal (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8′-carotenal Retinal (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal Not detected 3-OH-retinal 3R-3-OH-retinal 0 (3R)-3-OH-β-apo-8′-carotenal (3R)-3-OH-β-apo-12 -carotenal (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8′-carotenal 3-OH-retinal 3R-3-OH-retinal 3R-3-OH-retinal 3-OH-retinal Apo-8′-lycopenal (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal 3-OH-retinal 3-OH-retinal 3R-3-OH-retinal 3R-3-OH-retinal Apo-8′-lycopenal (3R)-3-OH-β-apo-12′-carotenal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-12′-carotenal 0 -lycopenal Apo-8 Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal (3R)-3-OH-β-apo-12′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-12′-carotenal Acycloretinal (slow) Acycloretinal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal 3-OH-retinal 3-OH-retinal 3R-3-OH-retinal 3R-3-OH-retinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal 3-OH-retinal 3R-3-OH-retinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal 0 -lycopenal Apo-8′-lycopenal Apo-10 No information Acycloretinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal Acycloretinal (slow) No information Acycloretinal Acycloretinal No information Acycloretinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal Apo-8′-lycopenal No information Acycloretinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal β-apo-8′-carotenol No information Acycloretinal Apo-10′-lycopenal Apo-10′-lycopenal 0 -carotenol Acycloretinal (slow) Acycloretinal β-apo-8 Int. J. Mol. Sci. 2016, 17, 1781 6 of 38 β-apo-8′-carotenol No information Acycloretinal No information Acycloretinal Apo-10′-lycopenal Int. J. Mol. Sci. 2016, 17, 1781 6 of Retinal 38 Retinal Retinal Retinal β-apo-8′-carotenol No information Acycloretinal Apo-10′-lycopenal Int. J. Mol. Sci. 2016, 17, 1781 6 of 38 Retinal Retinal β-apo-8′-carotenol β-apo-8′-carotenol No information Acycloretinal Apo-10′-lycopenal Retinal Retinal β-apo-8′-carotenol Retinal 0 -carotenol No information Retinal Acycloretinal (3R)-3-OH-β-apo-8 Retinal Retinal (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal β-apo-8′-carotenol No information Acycloretinal 3-OH-retinal 3-OH-retinal (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal Retinal Retinal β-apo-8′-carotenol (3R)-3-OH-β-apo-8′-carotenol Apo-8′-lycopenol (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal Retinal Retinal β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal 3-OH-retinal Apo-8′-lycopenol (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal Retinal Retinal 0 -lycopenol β-apo-8′-carotenol Apo-8 No information Acycloretinal Apo-8′-lycopenol (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal Retinal Retinal No information Acycloretinal No information Acycloretinal Apo-8′-lycopenol (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal Retinal Retinal No information No information Acycloretinal Acycloretinal (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal 4-oxo-β-apo-8′-carotenal 0 -carotenal 4-oxo-β-apo-8 (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal 4-oxo-β-apo-8′-carotenal 4-oxo-β-apo-8′-carotenal 4-oxo-retinal (slow)(slow) Not tested 4-oxo-retinal Not tested (3R)-3-OH-β-apo-8′-carotenol 3-OH-retinal 3-OH-retinal 4-oxo-β-apo-8′-carotenal

4-oxo-retinal (slow)

References References References

4-oxo-retinal (slow) 4-oxo-retinal (slow) [29] [29] [29]

Not tested Not tested [32]

Not tested [32] [32]

0 double bond, Enzyme cleaves C15–C15′ bond,positions. or equivalent equivalent positions. at thedouble C15–C15 or References [29] [32] positions. : Enzyme: cleaves at the C15–C15′ bond,double or equivalent

References : Enzyme cleaves at the C15–C15′ double [29] bond, or equivalent positions. [32]

2.2.2. CCDs Symmetrical Mode :with Enzyme cleaves at of theAction C15–C15′ 2.2.2. with CCDs Symmetrical Mode of double Actionbond, or equivalent positions. 2.2.2. CCDs with Symmetrical Mode of Action A β-carotene oxygenase from Microcystis, not identified is able toyet, carry two β-carotene oxygenase Microcystis, notyet, identified isout able to symmetrical carry out two symmetrical 2.2.2. CCDsA with Symmetrical Mode offrom Action cleavages on β-carotene and from zeaxanthin at thenot C7–C8 and C7–C8 C7′–C8′ double bonds [34].symmetrical It has been[34]. It has been Acleavages β-carotene oxygenase Microcystis, identified yet, is able toC7′–C8′ carry outdouble two on β-carotene and zeaxanthin at the and bonds A β-carotene from Microcystis, identified yet, able to carry out two suggested thatβ-carotene oneoxygenase molecule crocetindial two molecules ofis β-cyclocitral are released each cleavages on andof zeaxanthin atand thenot C7–C8 and C7′–C8′ double bonds [34].symmetrical Itfrom has been suggested that one molecule of crocetindial and two molecules of β-cyclocitral are released from each cleavages of on β-carotene andof zeaxanthin the two C7–C8 and C7′–C8′ double bonds [34].molecules Itfrom has been molecule β-carotene [19]. On the otherathand, onemolecules molecule ofβ-cyclocitral crocetindial andreleased two of suggested that one molecule crocetindial and of are each molecule ofmolecule β-carotene [19]. On the hand, one molecule ofare crocetindial and two molecules of suggested that one andother of from each hydroxyl-β-cyclocitral would becrocetindial released atwo molecule of zeaxanthin. molecule of β-carotene [19]. of On the otherfrom hand, onemolecules molecule ofβ-cyclocitral crocetindial andreleased two molecules of hydroxyl-β-cyclocitral would befrom released a molecule of zeaxanthin. molecule of β-carotene [19]. On the other hand, onefrom molecule of crocetindial andbacterial two molecules of Carotenoid oxygenases are responsible the generation of some aromatic hydroxyl-β-cyclocitral would be released a for molecule of zeaxanthin. hydroxyl-β-cyclocitral would be released from a for molecule compounds, as those produced by cyanobacterial species of zeaxanthin. the generaofCalotrix Plectonema. For Carotenoid oxygenases are responsible the generation some and bacterial aromatic

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2.2.2. CCDs with Symmetrical Mode of Action A β-carotene oxygenase from Microcystis, not identified yet, is able to carry out two symmetrical cleavages on β-carotene and zeaxanthin at the C7–C8 and C70 –C80 double bonds [34]. It has been suggested that one molecule of crocetindial and two molecules ofβ-cyclocitral are released from each molecule of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. Carotenoid oxygenases are responsible for the generation of some bacterial aromatic compounds, as those produced by cyanobacterial species of the genera Calotrix and Plectonema. For instance, Plectonema notatum PCC 6306 and Plectonema sp. PCC 7410 produced 6-methyl-5-hepten-2-one, β-ionone, 2,6,6-trimethylcyclohexanone, β-cyclocitral, 2-hydroxy-2,6,6-trimethylcyclo-hexan-1-one, 6-methyl-5-hepten-2-ol, dihydroactinidiolide, and β-ionone-5,6-epoxide. Moreover, Plectonema notatum PCC 6306 and Plectonema sp. PCC 7410 produced cyclogeraniol, 4-oxo-β-ionone, and dihydro-β-ionol. Based on Microcystis data, carotenoid oxygenases may be also present in biofilms with Phormidium sp., Rivularia sp., and Tolypothrix distorta [35]. 2.2.3. Carotenoid Oxygenases Cleaving at Different Positions NSC1, also known as NosCCD, is a soluble CCD from Nostoc sp. PCC 7120 that shares 44% homology with AtCCD1 from Arabidopsis thaliana, but only 26% identity with NSC3, also from Nostoc sp. When purified, NSC1 was incubated with β-apo-80 -carotenal in vitro, HPLC chromatograms showed a peak corresponding to 8,100 -apocarotenal, and a second product was identified by GC-MS as the volatile β-ionone [33]. If the incubation was kept for 5 h, the product 8,100 -apocarotenal was not accumulated proportionally to the disappearance of the substrate. Moreover, when the incubation was prolonged to 14 h, the 8,100 -apocarotenal disappeared, and isomers of 8,100 -apocarotenal and additional products resulting from the cleavage of β-apo-80 -carotenal at its C7–C8 and C90 –C100 bonds, were identified [33]. In any case, the main target were the C9–C10 double bonds and cleavage at other positions releases only minor products that may be a mechanism of eliminating competing species. In summary, in vitro NSC1 cleaves at C9–C10 and C90 –C100 double bonds in bicyclic carotenoids and at C9–C10 and C70 –C80 double bonds in monocyclic carotenoids. Interestingly, β,β-carotene cleavage products such as β-ionone, methylheptenone, and geranylacetone are known to inhibit growth of some cyanobacteria [35]. NSC3, also named NosDiox2, cleaves β-apo-80 -carotenals at C13–C14, C13 0 –C140 , and C15–C150 double bonds in vitro; in vivo, NSC3 cleaved C 30 compounds, such as 4,4 0 -diaponeurosporene at the C13–C140 double bond and 4,40 -diaponeurosporen-40 -al at the C90 –C100 double bond. Using C40 carotenoids as substrate, NSC3 cleaved torulene at C15–C150 double bond and seemingly also 3,4,30 ,40 -tetrahydrolycopene, but the cleaving site was not identified in that case; however, no products were detected using lycopene [31]. The explanation could be that the fully conjugated double bonds of the carotenoid backbone could facilitate the entry and the binding to the active pocket of the enzyme [23]. In vitro, 4,40 -diaponeurosporene and 4,40 -diaponeurosporen-40 -al were cleaved at more sites than in vivo. Discrepancies were observed in torulene cleavage by NSC3, which might be due to the differences among Escherichia coli strains and protein solubilisation [33]. Apocarotenals and apocarotendials were shown to have anticancer effects, suggesting that NSC3 could be used biotechnologically to produce novel bioactive compounds [31]. Several mycobacterial species are known to synthesize carotenoids; however, Mycobacterium tuberculosis does not contain carotenogenic genes, which were probably lost during evolution. Nevertheless, two ORFs coding for putative carotenoid cleavage oxygenases, Rv0654 and Rv0913c, were found in the genome of this species. Rv0654, named MtCCO from M. tuberculosis carotenoid cleavage oxygenase [36], shows a 44% similarity with the Nostoc CCO [30] and contains the four conserved His residues involved in binding the Fe2+ cofactor. Expression of MtCCO in E. coli as a GST fusion protein allowed its biochemical characterization in vitro. β-Apo-100 -carotenal was mainly cleaved by this enzyme at the C13–C14 double bond and

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less frequently at the C15–C150 double bond, while 3-OH-β-apo-11-carotenal was equally cleaved at both double bonds. MtCCO cleaved also C30 compounds efficiently, but exhibited only a weak activity on C25 compounds and no activity on shorter molecules, indicating that the substrate of this enzyme must have a minimal length of 25 carbons [36]. MtCCO also showed higher affinity for unsubstituted apocarotenoids, but the conversion was faster on those hydroxylated. Additionally, the enzyme cleaved C40 carotenoid substrates both symmetrically at the C15–C15 0 and asymmetrically at the C13–C14 or C130 –C140 double bonds, albeit the symmetrical and the asymmetrical cleavages were not equally targeted among the tested substrates (β-carotene, zeaxanthin, and lutein). For instance, there was a preference for a symmetrical cleavage when the β-ionone ring has a 3-OH radical (see zeaxanthin and lutein in Table 2). On the other hand, the acyclic lycopene was not cleaved by MtCCO in vitro, but it was converted to apo-13-lycopenone and apo-150 -lycopenal (acycloretinal) in lycopene-accumulating E. coli cells [36]. The enzyme NACOX1 from Novosphingobium aromaticivoransi exhibited cleavage activity at the C13–C14 double bound of carotenoids with a β-ionone ring giving β-apo-13-carotenone as resulting product [37]. Blast searches with the Nostoc NosCCD sequence in the genomes of the marine proteobacteria Sphingopyxis alaskensis and Plesiocystis pacifica retrieved two putative CCD genes whose identities with NosCCD ranged from 27% to 38%. After expression in carotenoid-expressing E. coli, only one of the two enzymes encoded by the putative CCD genes from both species, named respectively SaCCO and PpCCO, exhibited carotenoid cleavage activity [38]. Purified SaCCO cleaved apo-80 -carotenal in vitro and released apo-120 -carotenal and apo-100 -carotenal (Table 2); however, lycopene was poorly cleaved, and no activity was found against β-carotene or zeaxanthin. On the other hand, PpCCO cleaved zeaxanthin at the C130 –C140 double bond, producing apo-130 -zeaxanthinone and apo-140 -zeaxanthinal, and at the C110 –C120 double bond, releasing apo-120 -zeaxanthinal (Table 2). Similar products were observed after cantaxanthin, astaxanthin, and nostoxanthin were cleaved by PpCCO at the C130 –C140 position, producing apo-130 -cantaxanthinone and apo-140 -cantaxanthinal (Table 2), apo-130 -astaxanthinone and apo-140 -astaxanthinal (Table 2), and apo-140 -nostoxanthinal (Table 3), respectively. When PpCCO cleaves cantaxanthin and nostoxanthin at their C110 –C120 bonds, apo-120 -cantaxanthinal (Table 2) and apo-120 -nostoxanthinal (Table 3) are released, respectively.

8,10′-apocarotene 3-OH-β-apo-13-carotenone β-apo-13-carotenone (observed )( ) 3-OH-retinal 3R-3-OH-retinal and diapocarotenedial [32,33] (Table β-ionone ( ) + Apo-10,10′ β-apo-13-carotenone ( on ) β-carotene, ( ) zeaxanthin, torulene, lycopene, 3-OH-retinal 3R-3-OH-retinal Nostoc sp. PCC 7120 1). 3-OH-retinal ( ) 5 of 37 Not detected -dial ( ) -apocarotene-dial ( ) ( ) +products 3-OH-retinal ) Retinal3-OH-β-apo-13-carotenone ( β-apo-13-carotenone )( Retinal carotenone Other minority ( ) 8,10′-apocarotene ( )β-apo-14′-carotenal ( )Apo-8′-lycopenal Apo-10,10′ Assayed Substrates β-carotene-oxygenase (NSC1) MtCCO NSC3 Nostoc sp. NACOX1( ) Apo-12′SaCCOApo-10′Sphingopyxis PpCCO Plesiocystis -dial( (β-ionone ) Apo-8,10′ Retinal Retinal β-apo-4′-carotenal β-ionone ) +NosCCD 3-OH-retinal (Mycobacterium ) a β-apo-13-carotenone 10,10′-apocarotene 3-OH-β-apo-10′-carotenal and dialdehyde product (between C 5 to C15 ) (Table 1). However, larger substrates, such as 3-OH-β-ionone ( ) + Apo( , ) carotenal ( ) β-apo-8′-carotenal Transretinal ( ) -dial ( ) -apocarotene-dial ( ) 3-OH-retinal () ) 1. ( Enzymatic Retinal Microcystis 3-OH-β-ionone PCC -apocarotene-dial 7806 PCC 7120activity tuberculosis Novosphingobium pacifica 3-OH-β-apo-13-carotenone ( ) (3R)-3-OH-β-apo-12′-carotenal β-apo-13-carotenone ( ) (Table ) of bacterial apo-carotenoid Diox1 and NosAco γ-carotene β-apo-13carotenal ( alaskensis ) Retinalcleavage (sp. ) PCC (Nostoc ) + ApoNot detected 3-OH-β-apo-8′-carotenal Acycloretinal (slow) oxygenases Acycloretinal -dial ( 7120 ) specificity Apo-8′-lycopenal Enzymes and(3R)-3-OH-β-apo-8′-carotenal Products 10,10′-apocarotene Table 2. Enzimatic activity of different bacterial carotenoid oxygenases. 3-OH-β-apo-13-carotenone ( ) and β-apo-10′-carotenal 3-OH-β-apo-10′-carotenal a 40 dialdehyde product (between C 5 to C15) (Table 1). However, larger substrates, such as3-OH-retinal ( ) β-ionone ( β-ionone ) +and Apo-8,10′ β-apo-133-OH-β-ionone ( substrate ) + Apo(C ), are not cleaved [29]. β-apo-13-carotenone Apo-12′Apo-8,10′ carotenone ( ) Other minority products 8,10′-apocarotene ( ) β-carotene β-apo-13-carotenone ( ) Nostoc (showing ) β-apo-14′-carotenal β-ionone (( ))++Apo-10,10′ 3-OH-retinal 3R-3-OH-retinal γ-carotene β-apo-12′-carotenal 3-OH-β-ionone ( 3-OH-β-apo-13-carotenone ) MtCCO +3-OH-retinal Apo-Retinal 3-OH-β-apo-8′-carotenal Assayed Substrates Acycloretinal Acycloretinal products different substrates. β-carotene-oxygenase NosCCD (NSC1) Mycobacterium NSC3 sp. tocarotenone NACOX1 SaCCOApo-10′Sphingopyxis PpCCO Plesiocystis β-apo-8′-carotenal -dial(slow) ( ) ( ) -apocarotene-dial ( ) ( ) 10,10′-apocarotene 3-OH-retinal 3R-3-OH-retinal 3-OH-β-apo-13-carotenone ( ) β-apo-13-carotenone ( ) ( ) β-apo-13carotenal ( ) -apocarotene-dial ( ) β-ionone ( ) + Apo-8,10′ β-apo-13β-carotene (C 40 ), are not cleaved [29]. , PCC ) -dial(sp. ( -apocarotene-dial ) ) 8,10′-apocarotene ( ) named also NSC2 [30], iscarotenal one of( the three CCDs ( ) 3-OH-retinal Retinal ) Transretinal ( ) ( NosACO, Microcystis PCC 7806 Nostoc PCC 7120 tuberculosis Novosphingobium alaskensis pacifica of the cyanobacterium β-apo-8′-carotenal Retinal Not detectedNostoc sp. -dial ( minority )7120 products β-apo-14′-carotenal 3-OH-retinal ( Products ) Apo-10′-lycopenal Enzymes and carotenone ( () )γ-carotene Apo-10′Other -apocarotene-dial ( ) carotenone Retinal ( ) 3-OH-β-apo-10′-carotenal β-apo-10′-carotenal 3-OH-β-ionone ( ) + Apoβ-ionone ( ) + Apo-8,10′ β-apo-13-dial ( ) β-apo-13-carotenone ((strain β-carotene Diox1 NosAco β-apo-13-carotenone NosACO, also named NSC2 [30], is No one ofPpCCO the Plesiocystis three CCDs cyanobacterium Nostoc sp. Apo-12′β-ionone(3-OH-β-ionone ( ))++Apo-10,10′ Apo-8,10′ Retinal Retinal Int. J. Mol. Sci. 2016, 17, 1781 8 ofpreferences, 37 of the PCC 7120). The three enzymes exhibit different as expected from β-apo-13-carotenone () ) β-ionone information Acycloretinal ) MtCCO + Apo3-OH-β-apo-8′-carotenal γ-carotene Assayed Substrates ( , ()3-OH-β-apo-13-carotenone 2β-carotene-oxygenase × β-cyclocitral ( , ) Apo-10,10′-apocarotene carotenal ( ) substrate Apo-10′-lycopenal NosCCD (NSC1) Mycobacterium NSC3 Nostoc sp.Substrates SaCCO Sphingopyxis Apo-8′-lycopenal β-apo-8′-carotenal ( NACOX1 ) 10,10′-apocarotene -apocarotene-dial ( ) carotenone ( 3-OH-β-apo-13-carotenone )( )Transretinal 3-OH-β-apo-10′-carotenal (3R)-3-OH-β-apo-12′-carotenal No No (in vitro) NoPCC 6803 ( ) Retinal ( β-apo-13-carotenone β-apo-13carotenal ( ) -apocarotene-dial ( ) β-apo-13-carotenone +3-OH-retinal Apoβ-ionone (3-OH-β-ionone ) + Apo-8,10′ β-apo-13( ( No )(functions β-carotene Microcystis Synechocystis sp. Nostoc sp. PCC 7120 -apocarotene-dial (( )) Retinal ( ) ) cooperative 8,10′-apocarotene (strain PCC 7120). The three[31]. enzymes exhibit different substrate preferences, as expected from Acycloretinal CrocetindialPCC ) -dial (3R)-3-OH-β-apo-8′-carotenal 7806 Nostoc sp. 7120 PCC 7120 3-OH-β-apo-13-carotenone ) tuberculosis Novosphingobium alaskensis pacifica among them In vitro, NosACO cleaves monocyclic or carotenoids β-apo-10′-carotenal Noacyclic information Acycloretinal (slow) Acycloretinal 2( × ,β-cyclocitral ( -apocarotene-dial ,-dial ) ((PCC Apo-10,10′-apocarotene )) 10,10′-apocarotene 3-OH-retinal ( ) β-apo-14′-carotenal β-apo-14′-carotenal ( ) carotenone ( ) Apo-10′Other minority products ( ) carotenone ( ) Retinal ( ) β-apo-13-carotenone ( ) β-ionone-dial ( ) +( Apo-10,10′ No No Apo-12′No3-OH-retinal (in vitro) No 3R-3-OH-retinal Retinal (β-apo-13-carotenone ) β-carotene β-apo-4′-carotenal β-ionone 3-OH-retinal ( )bacterial ) β-apo-10′-carotenal TableCrocetindial 2. Enzimatic and of different carotenoid oxygenases. ( , activity )( ( ) +, Apo-8,10′ -dial 3R-3-OH-retinal cooperative among themcarotenal [31]. In( vitro, NosACO cleaves monocyclic or acyclic β-apo-8′-carotenol γ-carotene 3-OH-β-ionone () ) + substrate Apo-(( )) ( specificity at C15–C15′ to generate retinal. In vivo, bleaching activity wascarotenoids also β-apo-13-carotenone ( ) 3-OH-β-apo-8′-carotenal 2 × β-cyclocitral ( , of ) NosACO Apo-10,10′-apocarotene ) 3-OH-retinal -dial β-apo-8′-carotenal Apo-13′Transretinal ( functions ) bonds -apocarotene-dial ) Retinal β-apo-14′-carotenal ( () double 3-OH-β-apo-10′-carotenal β-apo-13-carotenone ( ) ( (( ) ) ) ) No No ( )3-OH-β-apo-13-carotenone + Apoβ-ionone ( 3-OH-β-ionone ) + Apo-8,10′ β-apo-13Retinal ( ) β-apo-13carotenal ( ) -apocarotene-dial ( ) Retinal Crocetindial Not detected Retinal Retinal β-apo-13-carotenone β-carotene 8,10′-apocarotene 3-OH-β-apo-13-carotenone ( Apo-10′-lycopenal ) 3-OH-β-apo-13-carotenone ( ) at C15–C15′ ( , activity ) Retinal -dial ( ) zeaxanthinone ( ) Apo-13′hydroxi-β- ( , ) Retinal β-apo-10′-carotenal β-apo-8′-carotenol γ-carotene double bonds to generate retinal. In vivo, bleaching of NosACO was also 22××β-cyclocitral Apo-10,10′-apocarotene observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1). Zeaxanthin 3-OH-retinal ( ) β-apo-14′-carotenal Int. J. Mol. Sci. 2016, 17, 1781 8 of 37 10,10′-apocarotene 3-OH-β-ionone ( ) -apocarotene-dial ( ) carotenone ( ) β-apo-14′-carotenal ( ) carotenone ( ) β-apo-13-Apo-10′Other minority Retinal ( () ) (( )) β-apo-13-carotenone β-ionone (3-OH-β-ionone )β-ionone +(products Apo-10,10′ No No No (in vitro) No ) + Apo( )( + Apo-8,10′ 3-OH-β-apo-8′-carotenal Retinal -dial Apo-14′cyclocitral ( 2 ×, hydroxi-β3-OH-retinal ( and ) (3R)-3-OH-β-apo-12′-carotenal Enzymes Products 3-OH-β-apo-13-carotenone ( No 3-OH-β-apo-13-carotenone () ) Apo-8′-lycopenal Crocetindial ) Apo-10,10′-apocarotene ( ) )-dial ( )3-OH-β-apo-14′-carotenal zeaxanthinone ( Acycloretinal ) Retinal Retinal No information No No (in vitro) ( -dial , ) -apocarotene-dial carotenal ( ) Transretinal ( ) observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1). -apocarotene-dial ( ) Zeaxanthin (( )) ( ) 8,10′-apocarotene (MtCCO ) ) Retinal (3-OH-retinal ( ) Sphingopyxis β-apo-14′-carotenal 3-OH-β-apo-10′-carotenal zeaxanthinal ( ) Apo-14′( , ) 3-OH-β-ionone (3-OH-β-ionone ) + Apo- β-apo-13-carotenone β-apo-12′-carotenal Acycloretinal (slow) Acycloretinal Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) Mycobacterium sp. NACOX1carotenone SaCCO PpCCO Plesiocystis ( ) ( )( NSC3 3-OH-β-apo-14′-carotenal ( Nostoc ) β-apo-8′-carotenal β-carotene cyclocitral 3-OH-retinal ) -dial ( Apo-10,10′-apocarotene ) 3-OH-β-apo-13-carotenone 3-OH-β-apo-13-carotenone ( ) 2 × hydroxi-β- 3R-3-OH-retinal β-apo-10′-carotenal 2 ×Crocetindial β-cyclocitral( ( , , Table Apo-10,10′-apocarotene -dial ( and ) 3-OH-β-apo-11-carotenal No activity No No3-OH-retinal (inRetinal vitro) Apo-13′Apo-12′) )( , β-ionone Zeaxanthin 1. Enzymatic of β-apo-13bacterial apo-carotenoid cleavage oxygenases and NosAco Retinal Int. J. Mol. Sci. 2016, 17,γ-carotene 1781 8 of 37 10,10′-apocarotene 3-OH-β-ionone ( ) 2.) Nostoc Enzimatic substrate specificity of(3R)-3-OH-β-apo-8′-carotenol different bacterial carotenoid oxygenases. Microcystis PCC 7806 PCC PCC tuberculosis Novosphingobium alaskensis pacifica β-apo-13-carotenone ( )? Table Retinal detected ( sp. )β-ionone + Apo-10,10′ No No (in vitro) No zeaxanthinal ( Not ) Diox1 ) + Apo-8,10′ Retinal ( β-apo-13-carotenone ) 3-OH-retinal 3-OH-β-ionone ( activity )7120 +( Apo3-OH-β-apo-8′-carotenal ) 7120 3-OH-β-apo-14′-carotenal ( ) β-carotene Crocetindial cyclocitral 3-OH-retinal ( ) ( () )( )( No (2 ×,1781 ) -dial ( J. Apo-10,10′-apocarotene )Mol. -dial ( )3-OH-β-apo-13-carotenone )+3-OH-β-apo-13-carotenone 3-OH-β-apo-10′-carotenal zeaxanthinone ( ) 2 × hydroxi-β3-OH-retinal 3-OH-retinal β-apo-8′-carotenol (3R)-3-OH-β-apo-8′-carotenol 3-OH-β-ionone ( ApoInt. Sci. 2016, 17, 1781 9 of 37 zeaxanthinal ( ) Int. J. Mol. Sci. 2016, 17, 1781 9 ofApo-10,10′-apocarotene 37Diox19 of Int. J. Mol. Sci. 2016, 17, 37 NosAco β-cyclocitral ( , ) -dial ( ) No No β-apo-13-carotenone ( () ) β-ionone ) + Apo-8,10′ Apo-12′Crocetindial-apocarotene-dial ( , 8,10′-apocarotene ) ( -apocarotene-dial Zeaxanthin 1. Enzymatic activity of ( bacterial cleavage oxygenases and ( ) ( Retinal ( ) Retinal carotenone ) Apo-12′- apo-carotenoid β-apo-14′-carotenal ) 3-OH-β-apo-11-carotenal ?) products 3-OH-β-ionone 3-OH-β-apo-13-carotenone (Table showing to different substrates. No No No (in vitro) No ( , ) ( ) 3-OH-retinal ( ) Apo-10′-lycopenal β-apo-8′-carotenal 3-OH-β-apo-14′-carotenal Apo-14′cyclocitral 3-OH-retinal ( )( ) ( ) Lycopene Crocetindial ( -apocarotene-dial ,Apo-10,10′-apocarotene ) -dial (10,10′-apocarotene -dial ( ) -dial ( ) Retinal Retinal 3-OH-retinal 3-OH-retinal ? Yes (in vivo) β-apo-13-carotenone ( ) γ-carotene β-apo-13carotenal ( ) ( ) zeaxanthinal ( ) Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 Apo-8′-lycopenal ) No No No (in vitro) Apo-13′-Crocetindial ( , ) β-apo-10′-carotenal 3-OH-retinal ( (3R)-3-OH-β-apo-8′-carotenal )Enzymes and Products β-apo-14′-carotenal ( ) 3-OH-β-apo-11-carotenal Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases. No No poorly showing products to different substrates. No information Acycloretinal zeaxanthinal ( ) ( , ) β-ionone ( ) + Apo-8,10′ β-apo-133-OH-β-ionone ( ) + Apo3-OH-retinal ( ) 3-OH-β-apo-8′-carotenal -dial ( 3-OH-β-apo-13-carotenone ) β-apo-13-carotenone ( ) β-carotene Unknown product β-apo-14′-carotenal carotenone ( )NACOX1 Apo-10′Other products Int. J. Mol. Sci. 17,Sci. 1781 8 of 37(slow) (( )) Retinal ( ) Mycobacterium Lycopene 3-OH-β-apo-13-carotenone -dial ) zeaxanthinone ( )9Yes × hydroxi-βApo-12′3-OH-β-apo-10′-carotenal Diox1 NosAco Apo-12′Apo-8′-lycopenol (in vivo) ((NosCCD Int.2016, J. Mol. 2016,Assayed 17, 1781 of 37 Apo-12′Substrates 2 Crocetindial Acycloretinal Acycloretinal 2 ×β-carotene-oxygenase β-cyclocitral (3-OH-β-ionone , )minority Apo-10,10′-apocarotene MtCCO NSC3 Nostoc sp. SaCCO Sphingopyxis PpCCO Plesiocystis Retinal Retinal Apo-13′Zeaxanthin Apo-12′( , ) 3-OH-β-ionone ( ))+( Apo-apocarotene-dial ) (NSC1) carotenone ( ) NoNo 8,10′-apocarotene 3-OH-β-apo-11-carotenal 3-OH-β-apo-13-carotenone ( ?) Transretinal ( Substrates poorly 3-OH-retinal 3R-3-OH-retinal No (in vitro) Apo-14′No γ-carotene ( , Nostoc ) -dial carotenal (No )alaskensis )NoNo 3-OH-β-apo-14′-carotenal 3-OH-β-apo-13-carotenone cantaxanthinal ( ) cyclocitral cantaxanthinal ( ) sp. PCC ( )7120 3-OH-retinal ( Retinal ) ( ( )() ) (3R)-3-OH-β-apo-8′-carotenol Unknown product 10,10′-apocarotene Int. J. Mol. Sci. 2016, 17, 1781 8zeaxanthinal of Crocetindial ( , 7806 ) -dial ) 7120 Microcystis PCC PCC 7120 Noand tuberculosis Novosphingobium 3-OH-β-apo-13-carotenone ( ) information Acycloretinal Lycopene zeaxanthinone (Nostoc )cantaxanthinal 2 × hydroxi-βEnzymes Products Synechocystis sp.37Diox1 PCC 6803 NosAco Apo-12′β-ionone ( sp. )(+PCC Apo-8,10′ β-apo-13( pacifica ) No No (inApo-8′-lycopenol vitro) Apo-10,10′-apocarotene ( ) 3-OH-β-ionone 3-OH-retinal ( ) Zeaxanthin Enzymes and Products β-apo-14′-carotenal ( ) ( ) No No Substrates β-apo-8′-carotenol β-apo-10′-carotenal zeaxanthinal ( ) Apo-14′( , ) 3-OH-β-ionone (carotenone ) Apo-13′3-OH-retinal ( )( )(( )) Lutein 3-OH-β-ionone ( ) 3-OH-β-apo-13-carotenone Apo-13′3-OH-β-ionone Astaxanthin Astaxanthin -dial ( ) specificity β-apo-13-carotenone 3-OH-β-apo-14′-carotenal ( ( )) β-apo-13-carotenone β-carotene cyclocitral Apo-12′( Astaxanthin ) + 3-OH-β-apo-14′-carotenal Apo-8,10′ cantaxanthinal ( ) ( β-apo-13-carotenone )of ( ) Int. J. Mol. Sci. 2016, 17, 1781 8 ofYes 37 -dial 2. Enzimatic and substrate different bacterial carotenoid oxygenases. NoApo-13′information Acycloretinal Lycopene ( ) 3-OH-β-apo-10′-carotenal Apo-12′3-OH-retinal Synechocystis sp.Apo-13′PCC 68033-OH-retinal Nostoc PCC 7120 β-ionone ( ( β-ionone )-apocarotene-dial +)(NSC1) Apo-10,10′ β-apo-4′-carotenal (in vivo) β-apo-12′-carotenal (Apo-10,10′-apocarotene ) + ApoInt. J.Table Mol.Crocetindial 2016, 17, 1781 9(sp. of) 37 Apo-10′-lycopenal Assayed Substrates 2Sci. × β-cyclocitral ( activity , ) 3-OH-β-ionone Apo-10,10′-apocarotene β-carotene-oxygenase NosCCD MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis No No No (in vitro) β-apo-8′-carotenal Assayed Substrates Apo-12′( , ) 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) 3-OH-β-apo-11-carotenal ? 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) 3-OH-retinal ( ) No 3-OH, 4-oxo-β-inone ( ) astaxanthinone 3-OH-β-apo-13-carotenone ( ) β-carotene-oxygenase NosCCD (NSC1) Nostoc MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis No No poorly Retinal Retinal β-apo-13-carotenone ( ) (3R)-3-OH-β-apo-12′-carotenal No No (in( vitro) 3-OH-β-apo-14′-carotenal ) zeaxanthinal ( ) ( , ) -apocarotene-dial β-apo-13(cantaxanthinal )of 37No product -apocarotene-dial ( ) Retinal Retinal ( 3-OH-retinal ) γ-carotene ( 4-oxo-β-apo-8′-carotenal )( )( No Lutein 3-OH-β-ionone ) carotenal Apo-13′Astaxanthin 3-OH-β-apo-13-carotenone ( ) ( ) Int. J. Mol. Sci. 2016, 17, 1781 8 ( ) ( ) Unknown 10,10′-apocarotene Retinal Not detected 3-OH-β-apo-13-carotenone ( ) Crocetindial ( , ) -dial ( ) Microcystis PCC 7806 Nostoc sp. PCC 7120 PCC 7120 Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases. -dial ( ) tuberculosis Novosphingobium alaskensis pacifica ( ) 2 × hydroxi-βNo information Acycloretinal β-apo-4′-carotenal β-ionone ( Other )3-OH-β-ionone + Apo-8,10′ β-apo-13-carotenone ( ) zeaxanthinalzeaxanthinone ( ) Apo-12′Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene Apo-14′β-apo-13-carotenone 3-OH-α-apo-15′-carotenal Apo-14′β-carotene Zeaxanthin 3-OH-retinal ( 3-OH-retinal ) ( ) ( tuberculosis Crocetindial ( Apo-10,10′-apocarotene )β-apo-14′-carotenal minority products 3-OH-β-apo-11-carotenal Retinal () ( )() )? β-apo-14′-carotenal No 7120Lutein 3-OH, 4-oxo-β-inone ( 3-OH-retinal ) Apo-10′Microcystis PCC 7806 PCC PCC Novosphingobium alaskensis pacifica 3-OH-β-apo-14′-carotenal ( ) astaxanthinone ( ) 3R-3-OH-retinal (( )) 7120 2 × β-cyclocitral ((β-ionone ,,3-OH-β-ionone )) ( sp. Apo-10,10′-apocarotene Astaxanthin -dial ( ) +) Apo-8,10′ 3-OH-β-apo-14′-carotenal Int. J. Mol. Sci. 2016, 17, Lycopene 1781 8 (slow) of Apo-13′37 Apo-14′cyclocitral β-apo-13-carotenone Apo-12′4-oxo-retinal Not tested Enzymes Products -apocarotene-dial carotenone ( ) No 4-oxo-β-apo-8′-carotenal Retinal Not( detected () ) astaxanthinal ( ) -dial3-OH-β-apo-13-carotenone ( of ) different astaxanthinal ( )Apo-12′Table 2. Enzimatic activity and substrate bacterial oxygenases. -dial ( )and ) No Apo-8′-lycopenol vivo) zeaxanthinal Retinal ( ( ))carotenoid ( )3-OH-α-apo-15′-carotenal 3-OH-β-ionone ( ) +specificity ( ) astaxanthinal No -dial No (in vitro) Apo-10,10′-apocarotene 3-OH-β-apo-8′-carotenal (Apocarotenal (Yes ) (in β-apo-8′-carotenal ( No Apo-13′Apo-10,10′-apocarotene Apo-14′3-OH, () (, ) )) 3-OH-β-apo-13-carotenone astaxanthinone ( ) Crocetindial , -apocarotene-dial ) 4-oxo-β-inone -dial 3-OH-retinal ( ) No( ) Transretinal poorlyPpCCO No (( )( ) ) sp. zeaxanthinal ( [38] ) cantaxanthinal ( , () (NSC1) (3R)-3-OH-β-apo-8′-carotenol β-apo-13-carotenone ( )Nostoc 3-OH-retinal 3-OH-β-apo-14′-carotenal γ-carotene β-apo-13carotenal ( ) ( 0 Lutein Assayed Substrates β-carotene-oxygenase NosCCD MtCCO Mycobacterium NSC3 NACOX1 SaCCO Sphingopyxis Plesiocystis β-apo-8′-carotenal References [19] [30,33] [36] [31] [37] [38] [38] References [19] [30,33] [36] [31] [37] [38] ( ) References [19] [30,33] [36] [31] [37] [38] [38] Unknown product 4-oxo-retinal (slow) Not tested Table 2. Enzimatic and substrate specificity of different bacterial oxygenases. β-apo-14′-carotenal ( (3R)-3-OH-β-apo-8′-carotenal ) Enzymes and Products 8,10′-apocarotene 4-oxo-β-ionone )) + (Apo-8,10 ) 3-OH-β-apo-13-carotenone ( )) carotenoid β-ionone (( -dial β-apo-13-carotenone No information Lycopene zeaxanthinone (Yes) (in vivo) Acycloretinal 2 × hydroxi-β-activity -dial ( ) astaxanthinal ( ) Int. J. Mol. Sci. 2016, 17, Echinenone 1781 8 of 37 β-apo-10′-carotenal β-apo-8′-carotenol ( β-ionone ( ) + Apo-8,10′ β-apo-130 Apo-10,10′-apocarotene Apo-14′β-apo-13-carotenone ( ) 3-OH-α-apo-15′-carotenal β-carotene Apo-12′Crocetindial ( , ) Zeaxanthin 3-OH-retinal β-apo-14′-carotenal 3-OH-β-apo-11-carotenal 3-OH-β-ionone ( ( ) )tuberculosis ( ) No Apo-10′- pacifica Other minority products β-apo-8 Retinal ( (NSC3 )(( )PCC 3-OH-retinal ) (Nostoc β-apo-13-carotenone ( ?) Apo-8′-lycopenal No carotenone poorly No alaskensis Microcystis PCC 7806 Nostoc sp.Apo-10,10′-apocarotene PCC 7120 7120 Novosphingobium β-ionone +3-OH-β-apo-13-carotenone Apo-10,10′ 3-OH-retinal 3-OH-retinal Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 Retinal Retinal 3-OH-β-ionone ) Apo-13′0 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene Astaxanthin Assayed -carotenal Substrates Apo-13′-dial ( ) β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis 3-OH-β-apo-14′-carotenal ) ( ) β-apo-8′-carotenal Apo-14′cyclocitral Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases. References [29] [32] References [19] [30,33] [36]zeaxanthinal [31] [38] [38] Unknown product Symbols indicate double bonds where oxygenases cleave. :(inC9–C10; :: C13–C14; : -carotenal C13′–C14′; ( [37] : C11′-C12′; (: C9′–C10′; -apocarotene-dial carotenone ( : ) C7–C8; and Products ( )) Enzymes Symbols indicate bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; C11′-C12′; C9′–C10′; Symbolsdouble indicate double bonds where cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; apocarotene-dial ( β-apo-13-carotenone ( ) ( ) β-apo-13Apo-12 ) 3-OH-retinal 3R-3-OH-retinal -dial ( )4-oxo-β-ionone ) ( ) oxygenases astaxanthinal ( ):: C15–C15′; Retinal Retinal No No β-ionone Retinal ( ) ) ( ) No No No vitro) Apo-10,10′-apocarotene ( , carotenal ( ) Transretinal ( ) Echinenone 3-OH-α-apo-15′-carotenal ( ) 4-oxo-β-inone β-apo-13-carotenone Retinal ) Apo-12′)PCC + Apo-8,10′ 3-OH, astaxanthinone ( ) 3-OH-β-apo-13-carotenone ( ) Novosphingobium Crocetindial , hydroxi-β) (sp.3-OH-β-ionone -dial ( )( ) )MtCCO zeaxanthinone ( )Acycloretinal ×NosCCD Microcystis PCC 7806 7120 -dial ( -apocarotene-dial tuberculosis alaskensis pacifica Int. J. Mol.Assayed Sci. 2016, 17, 1781 8 of 37 3-OH-β-apo-10′-carotenal 3-OH-β-apo-14′-carotenal )) ( 7120 zeaxanthinal ( ) ( [19] , ()2Nostoc 3-OH-retinal (NSC3 )((PCC + ApoLutein Acycloretinal (slow) Retinal Retinal 0 -carotenal 04-oxo-β-ionone Substrates Apo-10,10′-apocarotene β-carotene-oxygenase (NSC1) Mycobacterium NACOX1 SaCCO Sphingopyxis PpCCO β-apo-8′-carotenal Enzymes and 3-OH-β-apo-13-carotenone (bonds References [30,33] [36] indicate [31] [37] [38] [38]bold. Zeaxanthin [29] 3-OH-β-ionone ( the )β-apo-14′-carotenal (Products )Nostoc 4-oxo-β-apo-8′-carotenal Lycopene Symbols oxygenases cleave. :References C7–C8; : of C9–C10; C13–C14; :-carotenal C15–C15′; C13′–C14′;or information : [32] C11′-C12′; : C9′–C10′; -dial ( :at ) C7′–C8′. 3-OH-β-apo-13-carotenone When an cleaves atsp. different positions, the predominant isPlesiocystis indicated Lack ofor bold means no product Enzyme cleaves at the C15–C15′ double bond, or equivalent positions. Other products () :)oxygenases. carotenone ): vivo) Apo-10 (( )):preference When an enzyme cleaves at different predominant product is indicated in bold. Lack ofApo-12′bold no product preference or( (in information : C7′–C8′. When an enzyme cleaves different positions, predominant product iswhere indicated inβ-apo-14 bold. Lack bold means no preference information Int. J. Mol. Sci. 2016, 17, 1781 8product 37in Yes β-apo-10′-carotenal Table 2. Enzimatic specificity of different carotenoid β-apo-13-carotenone ( enzyme ) ( the (( )double )Retinal ) bacterial β-apo-13carotenal ( means ) ofproduct (substrate ) minority Apo-10,10′-apocarotene Apo-14′3-OH-β-apo-14′-carotenal Echinenone β-carotene : C7′–C8′. cyclocitral β-ionone ) positions, Apo-12′- Apo-14′Crocetindial (-apocarotene-dial ,activity ) No β-ionone ( )β-ionone +and Apo-8,10′ 10,10′-apocarotene 3-OH-retinal ( β-apo-13-carotenone ) ( Products No No poorly () )? 7120 sp.( ( ) )Novosphingobium -dialtuberculosis ((Mycobacterium )β-apo-13-carotenone Microcystis 7806 PCC 7120( 3-OH-β-ionone PCC alaskensis Apo-12′) +( Apo-10,10′ Apo-8′-lycopenol 3-OH-β-apo-14′-carotenal β-apo-10′-carotenal Substrates ) 3-OH-β-apo-11-carotenal +β-apo-13-carotenone Apoβ-carotene-oxygenase (NSC1) MtCCO NACOX1 Sphingopyxis PpCCO Plesiocystis n1781Mindicate S o Not tested 3-OH-β-apo-8′-carotenal Lutein No ( ) NoSaCCO No Nopacifica (in vitro)8: (slow) Apo-10,10′-apocarotene 2 bonds ×PCC β-cyclocitral ( Nostoc ,NosCCD ) sp.Apo-10,10′-apocarotene Enzymes and Apo-13′Apo-10,10′-apocarotene β-apo-8′-carotenal product 4-oxo-retinal 3-OH-retinal (NSC3 ) : Nostoc Int. J. Mol.Assayed Sci. 2016, 17,Canthaxantin of 37 Unknown β-apo-14′-carotenal Symbols double where oxygenases cleave. : C7–C8; C9–C10; C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; C9′–C10′; carotenone Apo-10′Other minority products 4-oxo-β-ionone ( ) 3-OH-β-apo-13-carotenone ( ) Retinal ( ) : Enzyme cleaves at the C15–C15′ double bond, or( equivalent positions. ( , ) Transretinal ( ) -dial ( ) astaxanthinal ( )bold : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of zeaxanthinal ( ) ( , ) not available. (3R)-3-OH-β-apo-8′-carotenol (3R)-3-OH-β-apo-12′-carotenal No No No (in vitro) No not available. 3-OH-retinal ( ) β-apo-13-carotenone ( ) ( ) not available. Retinal ( ) No zeaxanthinal ( ) β-apo-13carotenal ( ) -apocarotene-dial ( ) -dial ( ) 8,10′-apocarotene ) -dial Echinenone β-carotene-oxygenase β-ionone (3-OH-α-apo-15′-carotenal ) of different cantaxanthinal ) means no product preference or information -apocarotene-dial ( ) (tuberculosis Retinal ( β-apo-13-carotenone ) PCC Apo-12′+and Apo-8,10′ Microcystis PCC 7806 7120 7120 Novosphingobium alaskensis pacifica ) No information Acycloretinal 3-OH-retinal ( ) No 3-OH-β-apo-13-carotenone ( ) Crocetindial ( β-ionone ,Nostoc ) ((sp.,)PCC -dial ( Retinal Retinal zeaxanthinone ( ) 2 × hydroxi-βAssayed Substrates -dial ( ) NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis Table 2. Enzimatic activity substrate specificity bacterial carotenoid oxygenases. Apo-13′-cantaβ-apo-10′-carotenal carotenal ( ) Apo-10,10′-apocarotene β-apo-8′-carotenal 3-OH-β-apo-13-carotenone (? Apo-10′-lycopenal ) carotenone Transretinal )bold. 3-OH-retinal ( ( in )with References [19] (predominant [30,33] [36] [31]product [37]3-OH-retinal [38] [38] Apo-12′Crocetindial ( 2 positions, ) substrate Zeaxanthin β-apo-14′-carotenal 3-OH-β-ionone ( specificity β-apo-14′-carotenal (indicated ) ( CCDs ( ) means Apo-10′Other minority products x, )4-oxo-β-ionone )) Retinal Lycopene ( of )product Enzymes and Products : C7′–C8′. When an enzyme atβ-ionone different the is Lack of bold no preference orxanthinone information 3-OH-retinal 2.2.2. Symmetrical Mode of Action Yes (in vivo) Table 2.cleaves Enzimatic activity different bacterial oxygenases. 3R-3-OH-retinal (available. Canthaxantin γ-carotene not β-apo-13-carotenone ( 3-OH-β-apo-11-carotenal )) 3-OH-α-apo-15′-carotenal No Int. J. Mol. Sci. 2016, 17, 1781 9 (of )37 3-OH-β-ionone Apo-13′0 -carotenal Astaxanthin -dial ( ) 3-OH-β-apo-14′-carotenal β-apo-13carotenal ( ) poorly -apocarotene-dial ( ) β-apo-13-carotenone (( )7120 )) carotenoid Apo-12′(sp.)PCC +and Apo-8,10′ Apo-14′cyclocitral β-ionone Microcystis PCC 7806 Nostoc 7120 PCC tuberculosis Novosphingobium alaskensis ( )Apo-13′-cantaβ-apo-10 No No Retinal Retinal ) +v Nopacifica β-apo-10′-carotenal Lutein zeaxanthinal ( ) Acycloretinal (-dial ) + (Apo-8,10′ Apon M S o β-apo-8′-carotenal 3-OH-β-apo-8′-carotenal 0 -3-OH-β-apo-14′-carotenal Tab 2β-ionone Enz ma a β-apo-13-carotenone y and ub3-OH, a e peβ-apo-14′-carotenal y o (dsp.()e: )en ba e ( a):( C13–C14; a) β-apo-13o eno d oxygena e (in PpCCO NoReferences No vitro) Apo-10,10′-apocarotene ( eApo-10,10′-apocarotene ,3-OH-β-ionone )β-ionone carotenal ( ) No Apo-13′Transretinal Assayed Substrates [29]Unknown [32] β-carotene-oxygenase NosCCD (NSC1) MtCCO NSC3 NACOX1 SaCCO Sphingopyxis Plesiocystis product Table 2. Enzimatic activity oxygenases. astaxanthinone ( ) ))specificity Symbols indicate double bonds where oxygenases cleave. : ) C7–C8; C9–C10; : C15–C15′; : information C13′–C14′; : of : C9′–C10′; carotenone Apo-10′Other minority products + Apo-10,10 β-apo-13-carotenone 2 x(( 4-oxo-β-ionone (of3-OH-retinal 3-OH-β-apo-13-carotenone ( )4-oxo-β-inone 3-OH-β-apo-10′-carotenal Retinal )different ((( )) )carotenoid β-apo-13carotenal ( )with (substrate ) 3-OH-β-ionone zeaxanthinal (C11′-C12′; ) ( , β-ionone )-apocarotene-dial 2.2.2. CCDs Symmetrical Mode Action (( )) +4-oxo-β-ionone Apo-10,10′ not available. (Mycobacterium ) +(Apo( Nostoc (bacterial Canthaxantin Apo-14′β-apo-13-carotenone Apo-12′β-ionone +and Apo-8,10′ Echinenone β-ionone ( ) xanthinone ( ) β-apo-12′-carotenal 4-oxo-β-apo-8′-carotenal -apocarotene-dial ( ) carotenone ( ) 8,10′-apocarotene 3-OH-retinal ( ) No 3-OH-β-apo-13-carotenone ( ) Lycopene 3-OH-β-apo-13-carotenone ( ) -dial ( ) Enzymes and Products zeaxanthinone ( ) 2 × hydroxi-β-dial ( ) Yes (in vivo) β-apo-8′-carotenal β-apo-10′-carotenal Microcystis PCC 7806 Nostoc sp. PCC 7120 PCC 7120 tuberculosis Novosphingobium alaskensis pacifica yet, n M S o A β-carotene oxygenase from Microcystis, not identified is able to carry out two symmetrical Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene ( , ) carotenal ( ) Apo-10,10′-apocarotene 3-OH-β-apo-13-carotenone ( ) Transretinal ( ) β-apo-14′-carotenal carotenone ( ) Apo-10′Other minority products Zeaxanthin 3-OH-retinal ( ) Apo-12′Crocetindial ( , ) 10,10′-apocarotene Retinal ( ) 3-OH-β-ionone ( ) 3-OH-β-apo-11-carotenal ? β-apo-13-carotenone ( ) cantaxanthinal ( ) ( ) 4-oxo-β-ionone ( ) No No poorly : Enzyme cleaves at the C15–C15′ double bond, or equivalent positions. apocarotene-dial ( Retinal ( ) ) β-apo-13carotenal ( ) -apocarotene-dial ( ) 2 x 4-oxo-β-ionone ( ) Enzymes and Products : C7′–C8′. an enzyme cleaves different positions, the predominant product is indicatedSaCCO in bold. Lack of bold means no product preference or information Apo-12′Apo-8′-lycopenol β-ionone ( ) +(NSC1) Apo-10,10′ NoWhen Canthaxantin Apo-14′Apo-8′-lycopenal -dial ( at ) MtCCO 3-OH-α-apo-15′-carotenal Echinenone 3-OH-β-apo-14′-carotenal ( ) Assayed Substrates Retinal Not detected Apo-14′cyclocitral β-carotene-oxygenase NosCCD Mycobacterium NSC3 Nostoc sp. NACOX1 Sphingopyxis PpCCO Plesiocystis 3-OH-retinal ( ) 4-oxo-retinal (slow) Not tested Unknown product β-apo-12′-carotenal β-apo-13-carotenone Apo-12′β-ionone ( ) + Apo-8,10′ -dial (Transretinal astaxanthinal ( )is able to carry out two symmetrical ( ) and-dial -dial ) eβ-apo-13-carotenone 3-OH-β-apo-14′-carotenal ()Nostoc ) (on n) β-carotene m a carotenone nd duand Apo-13′-cantaβ-apo-10′-carotenal ( ma , (NSC1) ) products carotenal (at ) the Lutein zeaxanthinal (double ) ( ( Mycobacterium )a No NoSaCCO No (in vitro) Apo-10,10′-apocarotene Tab eOther 2-apocarotene-dial Enz a-dial ub y cleavages oand d)Products en ba eβ-apo-8′-carotenol a Po eno eβ-carotene ASphingopyxis oxygenase from Microcystis, not yet, β-apo-14′-carotenal ( e ( d) oxygena Apo-10′minority β-carotene Apo-10,10′-apocarotene Assayed Substrates ( pe ) Enzymes zeaxanthin C7–C8 and C7′–C8′ [34]. It has cantaxanthinal ( been ) β-carotene-oxygenase NosCCD MtCCO NSC3 sp. NACOX1 PpCCO Plesiocystis β-apo-8′-carotenal ( v ) yApo-10,10′-apocarotene ( ) identified No information Acycloretinal x ( Retinal ) 3-OH-β-apo-10′-carotenal (( )) [30,33] zeaxanthinal (cantaxanthinal ) [38] bonds ( References , 3-OH-β-ionone )β-ionone ) 7120 Microcystis PCC 7806 Nostoc sp.)3-OH-β-ionone PCC PCC tuberculosis Novosphingobium alaskensis pacifica (No +Apo-10,10′-apocarotene Apo-10,10′ not available. 3-OH-retinal ( ) (M (N )β-apo-13-carotenone +CCD ApoAcycloretinal (slow) Acycloretinal ( 2n )7120 +4-oxo-β-ionone Apo2 × β-cyclocitral ( , ) 3-OH-β-apo-8′-carotenal β-apo-13-carotenone ( ) β-apo-13carotenal ( ) -apocarotene-dial ( ) 3-OH-β-apo-13-carotenone ( ) β-ionone ( ) Retinal Not detected [19] [36] [31] [37] [38] xanthinone ( ) A d Sub n NSC M CCO m NSC N p NACOX S CCO PpCCO P 3-OH-retinal No 3-OH-β-apo-13-carotenone ( )d7120 ) (e sp. Lycopene γ-carotene -dial (a ) 3-OH-β-apo-13-carotenone β-apo-10′-carotenal ( Mycobacterium )J. Mol. ( )( (17, (sp., PCC ) Apo-10,10′-apocarotene Retinal Retinal carotenal (on -dial ( ) Yes (in No No No (in PpCCO vitro) pacifica Novivo) Transretinal ) ba Int. Sci. 2016, 9 of 37 Microcystis Crocetindial PCC 7806 7120 PCC Retinal ) y)1781 tuberculosis Novosphingobium alaskensis Assayed Substrates Apo-13′β-carotene-oxygenase NosCCD (NSC1) MtCCO NSC3 Nostoc SaCCO Sphingopyxis Plesiocystis Astaxanthin Tab ma y-dial and o(?CCDs en eNACOX1 a a (o )eno d oxygena e)and 0 -carotenal cleavages β-carotene and Apo-12′zeaxanthin at theare C7–C8 and C7′–C8′ double bonds [34]. It has been (β-ionone ,Nostoc ( Apo) β-ionone 10,10′-apocarotene 4-oxo-β-ionone ( )(() +a)) e3-OH-β-ionone 3-OH-β-apo-11-carotenal β-apo-13-carotenone ( pe ) Retinal suggested that molecule crocetindial two molecules β-cyclocitral released (ub 8,10′-apocarotene Crocetindial (-apocarotene-dial , )eminority )2(( Enz -dial (N v ) β-apo-13-carotenone Apo-12′β-ionone No poorly ))++Apo-10,10′ β-apo-14′-carotenal 3-OH-β-apo-8 carotenone Apo-10′Other products 3-OH-β-apo-14′-carotenal ( one ) NoSymmetrical 2.2.2. with Mode of Action Retinal (3-OH-retinal + Apo-8,10′ β-apo-13Canthaxantin Echinenone Lutein 3-OH-β-ionone (Apo-8,10′ )7120 +3-OH-β-ionone 3-OH-β-apo-8′-carotenal M PCC p )tuberculosis PCC PCC ( of Nalaskensis m ( )3-OH-α-apo-15′-carotenal References [29] ofApo-14′[32] from( each β-apo-8′-carotenal 3-OH-retinal ( ()n Unknown product (3-OH, ) cleave. β-apo-14′-carotenal ): m β-apo-10′-carotenal 4-oxo-β-inone ) nd(3R)-3-OH-β-apo-8′-carotenal ) double bonds oxygenases : C9–C10; : C13′–C14′; : C9′–C10′; MicrocystisSymbols PCC 7806 indicate Nostoc (sp. PCC 7120 β-apo-13-carotenone Novosphingobium pacifica ): C13–C14; Apo-12′- : ) C15–C15′; β-ionone )PCC Apo-8,10′ ( )3-OH-β-apo-13-carotenone ( where )β-ionone Apo-13′-cantadu zeaxanthinal ( ) : C11′-C12′; astaxanthinone Int. ((J. Mol. 17, 1781 9 of 37 (0 3-OH-β-apo-13-carotenone ) (β-apo-13-carotenone Apo-10,10′-apocarotene β-apo-13-carotenone ( (C7–C8; -dial (+Apo-8,10 β-apo-13-apocarotene-dial )-dial ) ) ( -dial carotenal )) 2016, β-apo-8′-carotenal Transretinal () β-carotene )P 4-oxo-β-apo-8′-carotenal -apocarotene-dial ) ) ((Sci. carotenone ( ) one β-ionone (( 2), +x Apo-10,10′ 3-OH-retinal () of No (that suggested molecule of Apo-13′crocetindial two molecules of β-cyclocitral are released from each 8,10′-apocarotene cantaxanthinal ( ) andand -apocarotene-dial 4-oxo-β-ionone ) Retinal molecule [19]. On the other hand, one molecule of crocetindial two molecules of A 3-OH-retinal A3-OH-β-apo-13-carotenone No ( ) Canthaxantin Apo-10′-lycopenal 3-OH-β-ionone ( ) + Apo3-OH-β-apo-8′-carotenal Apo-10,10′-apocarotene Apo-14′β-apo-13-carotenone ( ) ( (3R)-3-OH-β-apo-8′-carotenal 3R-3-OH-retinal β-apo-13-carotenone β-apo-13carotenal ( ) -apocarotene-dial ( ) ( ) Apo-12′β-ionone ( ) + Apo-8,10′ xanthinone )Apo-13′-canta() ) M( ) β-apo-14′-carotenal A Lycopene d Sub n 3-OH-β-ionone n minority N CCD NSC M CCO m NSC Nproduct NACOX S Lack CCO PpCCO Pis cleaves at the C15–C15′ double bond, or(able equivalent positions. 3-OH-retinal ( dup:) Enzyme -dial (Retinal ) Apo-12′: C7′–C8′. When anApo-10,10′-apocarotene enzyme at3-OH-retinal different the predominant is (indicated inApo-10′bold. of bold means no(inproduct preference or two information carotenone ) from Other 3-OH-β-apo-13-carotenone 3-OH-β-apo-10′-carotenal Yes vivo) γ-carotene n(3R)-3-OH-β-apo-8′-carotenol m nd P -apocarotene-dial ) cleaves A β-carotene oxygenase Microcystis, not identified yet, to carry out symmetrical β-apo-8′-carotenal β-apo-10′-carotenal 3-OH-α-apo-15′-carotenal (( ( positions, )) -dial ( ( products ) ) +( Apo4-oxo-retinal (slow) Not tested d 3-OH-β-apo-13-carotenone 4-oxo-β-ionone ( )) Retinal ( 8,10′-apocarotene No carotenone No poorly 2PCC xApo-8,10′ 4-oxo-β-ionone -dial ( ) ((( ))n n ( PCC astaxanthinal ( of) crocetindial9 and 3-OH-β-apo-13-carotenone (β-apo-13-carotenone ) phydroxyl-β-cyclocitral β-apo-14′-carotenal zeaxanthinone ( ) hand, one 2 × hydroxi-ββ-apo-13-carotenone ( ) ( ) Apo-10′Other minority products molecule of β-carotene [19]. On the other molecule two molecules of 3-OH-β-ionone ( ) + Apo3-OH-β-apo-14′-carotenal Int. J. Mol. Sci. 2016, 17, 1781 of 37 No information Acycloretinal 3-OH-β-apo-8′-carotenal Retinal ( β-apo-13carotenal ( ) -apocarotene-dial ( ) β-ionone ( ) + β-apo-13Lutein Apo-14′would be released from a molecule of zeaxanthin. β-ionone ( ) + Apo-10,10′ M PCC N p ) N m β-carotene Echinenone 3-OH-retinal 3R-3-OH-retinal β-ionone ( ) cantaxanthinal ( n, ) 3-OH-β-ionone carotenal ( ) S CCO 3-OH-retinal Zeaxanthin Unknown product ( ) NSC N xanthinone ( )3-OH-retinal9 of 37 ) CCO ( 3-OH-retinal ) ( ) MTransretinal A d SubInt. J. Mol.not n10,10′-apocarotene CCD NSC p NACOX PpCCO P ) 3-OH-β-apo-13-carotenone 3-OH-β-apo-10′-carotenal Apo-12′-( ) A and m d( (() M Sci. available. 2016, 17, 1781 + Apo23-OH-β-ionone ×Other β-cyclocitral (()-apocarotene-dial ) (NO Apo-10,10′-apocarotene R () () ) m 3-OH-β-apo-14′-carotenal -dial ( Apo-10,10′-apocarotene ) ,))-dial Apo-10,10′-apocarotene Apo-14′cyclocitral and C7–C8 bonds [34]. [38] Itofhas been References [36]No [31] [38] ( ,2016, carotenal (at) the β-apo-14′-carotenal 8,10′-apocarotene ) ( [30,33] ) β-carotene carotenone ( carotenone ) zeaxanthin Apo-10′minority ( 3-OH-retinal ) Retinal ) No cantaxanthinal ( double ) Apo-14′3-OH-retinal (Transretinal No ( Apo)1781 ( products 3-OH-β-apo-13-carotenone ) cleavages 3-OH-β-apo-8′-carotenal (in[37] vitro)C7′–C8′ No Canthaxantin A p[19] Retinal )(on J.-apocarotene-dial Mol. Sci. 9 of 37 Int. J. Mol. Sci. 2016, 17, 1781 9 of No 37would Apo-13′No3-OH-β-ionone 0 -carotenal hydroxyl-β-cyclocitral be released from a molecule zeaxanthin. No PCC No (3-OH-β-ionone β-apo-10′-carotenal MInt. PCC N PCC -dial (Apo-10,10′-apocarotene ) +17, N m NoA((in )vitro) 3-OH-retinal ( ) Astaxanthin ( )( CCDs (3R)-3-OH-β-apo-12′-carotenal ) cantaxanthinal ( ) 4-oxo-β-ionone 3-OH-β-apo-10 Crocetindial -dial ((3-OH-β-apo-13-carotenone )()( )) +3-OH-retinal T Symmetrical -dial ( 3-OH-β-apo-13-carotenone ) β-apo-13-carotenone with Mode of( Action -dial Apo-13′-cantazeaxanthinal ( ) ( , 3-OH-β-ionone ) 10,10′-apocarotene 3-OH-β-apo-10′-carotenal )( β-apo-14′-carotenal ) -dial carotenal )and β-ionone ((double ),((+ Apo-10,10′ (),) ) 3-OH-β-ionone +) bonds Apoβ-apo-10′-carotenal (that Echinenone 3-OH-α-apo-15′-carotenal 8,10′-apocarotene References [29] [32] p n(Transretinal n2.2.2. 3-OH-retinal ( )(( cleave. dwhere ( ) ) one 3-OH, 4-oxo-β-inone ( ) astaxanthinone suggested molecule of crocetindial two molecules of β-cyclocitral are released from each Symbols indicate oxygenases : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; cantaxanthinal ( ) 2 x 4-oxo-β-ionone ( ) 3-OH-β-apo-13-carotenone ( ) -dial ( ) Apo-12′3-OH-β-apo-13-carotenone ( ) β-apo-8′-carotenol 3-OH-β-apo-14′-carotenal ( ) A A Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 β-apo-13-carotenone ) Lutein γ-carotene 0 3-OH-β-ionone ( ) Apo-13′-( ) β-ionone ( ) (+Apo-10,10 Apo-10,10′ Apo-10,10′-apocarotene ( ) Astaxanthin ) ( Apoβ-apo-13-carotenone ( )? β-carotene 3-OH-β-ionone (-apocarotene-dial ) 3-OH-retinal 3-OH-β-apo-8′-carotenal (3R)-3-OH-β-apo-12′-carotenal ( ) 3-OH-retinal 3R-3-OH-retinal Apo-12′Crocetindial (-apocarotene-dial , No )-dial 3-OH-β-apo-11-carotenal ) Apo-10,10′-apocarotene Retinal ( ) R ( ( )) p β-apo-10′-carotenal Apo-12′) )) ++β-ionone 3-OH-β-ionone ((Apo-8,10′ ApoApo-8′-lycopenol 3-OH-β-apo-13-carotenone Int. 3-OH-β-apo-10′-carotenal J. Mol. Sci. 2016, 17, 1781 9 ofor37xanthinone A m d 3-OH-retinal β-ionone +Apo-10,10′-apocarotene β-apo-13Apo-10,10′-apocarotene Apo-14′2 × β-cyclocitral (10,10′-apocarotene , -dial )( )(O Apo-13′- positions. n cantaxanthinal ( ) A p( )((molecule n3-OH-retinal n n of d ( ) 3-OH-β-apo-13-carotenone 3-OH-retinal ) No 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) p n n : Enzyme cleaves at the C15–C15′ double bond, equivalent ( ) -apocarotene-dial ( ) -dial ( ) Apo-12′Apo-12′: C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product preference or information Retinal ( ) A Retinal Retinal β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of 8,10′-apocarotene No No No (in vitro) No Canthaxantin Retinal ) γ-carotene zeaxanthinal ) A β-carotene oxygenase from Microcystis, not yet,zeaxanthinone is( Apo-14′able to carry out two symmetrical ( ( 4-oxo-β-ionone ) ) +( Apo( ) 3-OH-retinal 3R-3-OH-retinal 3-OH-β-apo-10′-carotenal ( 3-OH-retinal )) ( d 3-OH-β-apo-13-carotenone ( ) carotenone ) ( ) (-dial ( ) -dial ( ) astaxanthinal ( ) Noidentified information Crocetindial (-apocarotene-dial ,× hydroxi-β)-dial -dial 3-OH-β-ionone T ( Acycloretinal ) cantaxanthinal 210,10′-apocarotene )d Apo-13′-canta3-OH-β-ionone (β-apo-13) Apo-13′R β-ionone ( )(+Astaxanthin Apo-8,10′ Apo-10,10′-apocarotene Apo-14′Echinenone 3-OH-β-ionone +O Apo- m β-ionone (( )) ( R cantaxanthinal ( ) 3-OH-β-apo-8′-carotenal cantaxanthinal ( A) 3-OH-β-apo-13-carotenone -dial 3-OH-β-ionone ( β-apo-14′-carotenal )3-OH-β-ionone () ) ( ) 2(d)x) )4-oxo-β-ionone (3-OH-retinal ) R( )3-OH-α-apo-15′-carotenal Lycopene Zeaxanthin Apo-12′Yes (in cantaxanthinal vivo) notAstaxanthin available. 3-OH-β-apo-13-carotenone ( )) hydroxyl-β-cyclocitral -dial ( Apo-10,10′-apocarotene ( cleavages Apo-13′would be released from a molecule of zeaxanthin. γ-carotene 3-OH-β-apo-14′-carotenal ( T ) β-carotene 10,10′-apocarotene Apo-14′cyclocitral 3-OH-β-ionone ( ) + ApoReferences [19] [30,33] [36] [31] [37] [38] [38] on and zeaxanthin at the C7–C8 and C7′–C8′ double bonds [34]. It has been 3-OH-β-apo-8′-carotenal xanthinone ( ) 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) -apocarotene-dial ( ) carotenone ( ) Apo-8′-lycopenal No No poorly -dial ( ) astaxanthinal ( ) Canthaxantin Apo-13′3-OH-β-ionone (8,10′-apocarotene ) ( Astaxanthin No3-OH-β-ionone 3-OH-β-apo-13-carotenone Astaxanthin No No Apo-13′No (inApo-12′vitro) Apo-10,10′-apocarotene 3-OH-β-apo-10′-carotenal β-ionone ) +Apo-10,10′-apocarotene 3-OH-retinal (( 4-oxo-β-inone )(( )() ) n n ( )3-OH-β-ionone ( ) β-apo-13Apo-13′(Apo-8,10′ ) + Apocantaxanthinal ( ) A) β-apo-13-carotenone β-carotene Ap Unknown product OH 3-OH, 3-OH-retinal ( 3-OH-β-apo-13-carotenone γ-carotene 2.2.2. CCDs with Symmetrical of[30,33] Action zeaxanthinal ( ) astaxanthinone ( )9 of 37 Apo-14′( -dial , )(( )) 8,10′-apocarotene ( Int.) (3R)-3-OH-β-apo-8′-carotenol (References ) 3-OH-retinal OH A-dialOH J.that Mol. Sci. 17, 1781ofMode OHγ-carotene Int. J. 3-OH, Mol.( Sci. 17, 1781 Apo-14′Apo-10,10′-apocarotene 0 -)) OH 2 × β-cyclocitral , 2016, ) Apo-10,10′-apocarotene -dial ( (slow) )ofApo-13′-canta[19] [36] [31] [37] [38] [38] 3-OH, ( β-apo-13)2016, astaxanthinone ) (Sci. )( 2016, astaxanthinone ( two ) cantaxanthinal Symbols indicate double bonds where cleave. : C7–C8; : C9–C10; :released C15–C15′; :each C13′–C14′; : C11′-C12′; : C9′–C10′; 4-oxo-β-apo-8′-carotenal suggested one molecule crocetindial molecules β-cyclocitral from Acycloretinal Acycloretinal ))-dial ( )3-OH-β-apo-13-carotenone ( Apo-10,10′-apocarotene -apocarotene-dial ( -dial ) 2 x( 4-oxo-β-ionone carotenone ( oxygenases ) (No ) 4-oxo-β-inone Apo-8′-lycopenal β-ionone +A Apo-8,10 β-apo-133-OH-retinal zeaxanthinone ( : )C13–C14; are 24-oxo-β-inone × hydroxi-ββ-ionone )((+4-oxo-β-ionone Apo-8,10′ Int. J.Crocetindial 17, 1781 9 (of 37 d()(OH No No (in vitro) No 3-OH-β-ionone ( Mol. )10,10′-apocarotene Apo-13′- and Astaxanthin Retinal ( ) () R Apo-14′Echinenone p n n β-apo-13-carotenone ( ) β-carotene β-ionone ( ) 3-OH-β-apo-13-carotenone ( ) Apo-12′, ) xanthinone ( ) -dial ) Zeaxanthin 3-OH-β-apo-11-carotenal ? 3-OH-β-ionone ( ) cantaxanthinal ( ) -dial ( ) astaxanthinal ( ) γ-carotene 3-OH-retinal ( ) ( ( , , ) ) 3-OH-β-ionone -dialApo-10,10′-apocarotene ( ) ) + Apo3-OH-retinal 3-OH-retinal OH Apo-10,10′-apocarotene n) Int. J. Mol. Sci. 2016, 17, 1781 2 ×Crocetindial 9 of 37 3-OH-β-apo-10′-carotenal ) Apo-13′Apo-14′Apo-10,10′-apocarotene Apo-14′Astaxanthin β-cyclocitral Apo-10,10′-apocarotene Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal (an Apo-14′cyclocitral -dial (+(Apo-8,10′ ) 17, Not tested ( )(1781 carotenone ( [19]. ) oxygenases 3-OH, 4-oxo-β-inone ( ) 3-OH-β-ionone astaxanthinone (of4-oxo-retinal ) C7–C8; Acycloretinal Acycloretinal d dOH β-apo-14′-carotenal ) ( When indicate where cleave. :37 :(slow) C9–C10; : C13–C14; :[38] C15–C15′; : of C13′–C14′; : C11′-C12′; : C9′–C10′; C7′–C8′. enzyme cleaves at different positions, the product is indicated in Lack ofmolecules bold means no product or information )2016, β-apo-13apocarotene-dial (n( :)Symbols carotenone ((slow) -dial ) molecule astaxanthinal (symmetrical ) of bonds β-carotene On the other hand, one molecule ofzeaxanthinal crocetindial and two 3-OH-β-apo-14′-carotenal ( NoA )double Int.β-ionone J.-apocarotene-dial Mol. Sci. 37 preference 3-OH-β-apo-13-carotenone No No (in vitro) No Int. J. Mol. Sci. 2016, 17, 1781 9predominant Canthaxantin No ( )bold. Apo-14′) ([19] β-carotene oxygenase from Microcystis, not identified out two 3-OH-β-apo-10′-carotenal d( 4-oxo-β-inone No No (in vitro) Apo-10,10′-apocarotene [30,33] [36] [31] [37]) to carry [38](9 of (References Apo( )( R) β-carotene A (Retinal OH Lutein 3-OH, ( )3-OH-β-apo-13-carotenone ) β-apo-13-carotenone (yet, ) is( able -dial )NoSci. 2016, astaxanthinal ) ) 10,10′-apocarotene astaxanthinal ( ) astaxanthinone ( ( , , -dial )( ) , ( 3-OH-β-ionone -dial ( OH ) ) +-dial -dial ) Apo-12′) Apo-13′-canta( ) ( ) Int. J. (Mol. 17, Apo-10,10′-apocarotene 2 Crocetindial × β-cyclocitral Apo-10,10′-apocarotene γ-carotene -apocarotene-dial ( -dial ) ( OH carotenone ( different )1781No positions, OH Lycopene Apo-13′- zeaxanthinal References [36] [31] Apo-14′[37] [38] A 3-OH-retinal cantaxanthinal ( in ) bold. [38] β-apo-14′-carotenal ()( [30,33] ))OH ( When Apo-10′-lycopenal 3-OH-retinal ) No ( ):available. ([19] ) β-apo-13-carotenone Apo-12′-no product preference or information 2Apo-8,10′ x 4-oxo-β-ionone (3-OH-retinal ) RetinalOH OH β-carotene C7′–C8′. an enzyme cleaves at the predominant product is indicated Lack of bold means 10,10′-apocarotene not Yes (in vivo) OH A Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 No No (in vitro) No hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. ( Apo-10,10′-apocarotene Apo-14′β-ionone ( ) + β-apo-13References [19] [30,33] [36] [31] [37] [38] [38] References [19] [30,33] [36] [31] [37] [38] [38] β-ionone ( ) cleavages on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ double bonds [34]. It has been canta cantaxanthinal ( ) -dial ( ) Apo-12′-( ): C13′–C14′; ( , Symbols )-dial ( ) indicate xanthinone double bonds where cleave. Apo-8′-lycopenol : C7–C8; : C9–C10; : C15–C15′; : C11′-C12′; : C9′–C10′; OH A 3-OH-β-apo-11-carotenal ? OH OH No No : C13–C14; poorly -dial astaxanthinal ( )zeaxanthinone 3-OH-retinal ( OH ) ( n) oxygenases 3-OH-β-apo-13-carotenone Crocetindial (Crocetindial , , )( ) ) Apo-10,10′-apocarotene Apo-12′( ) 2 × hydroxi-β2 × β-cyclocitral ( 3-OH-α-apo-15′-carotenal OH Apo-13′cantaxanthinal ( ) Apo-10,10′-apocarotene References [29] [32] -dial-dial ( bonds ) ( ( () )d product : Acycloretinal Zeaxanthin 3-OH-β-ionone Symbols[30,33] indicate double where oxygenases cleave. : C7–C8; :vitro) C15–C15′; :No C13′–C14′; : )C11′-C12′; C9′–C10′; β-apo-14′-carotenal ( (available. )) β-apo-13-carotenone ):( C13–C14; Apo-12′No(Apo-10′-lycopenal No (in Apo-12′Noinformation astaxanthinal ( ) Unknown -apocarotene-dial ) [36] ) Retinaln( not ) [31] OH n) [37] : C9–C10; β-apo-13-carotenone β-carotene 3-OH-β-ionone 3-OH-β-ionone ( No Apo-13′- from Astaxanthin zeaxanthinal ( C13′–C14′; )( ) Apo-14′Astaxanthin 03-OH-β-apo-14′-carotenal γ-caroteneCanthaxantin ( (oxygenases )) C15–C15′; References [38] [38] cantaxanthinal ( β-cyclocitral Apo-14′cyclocitral Symbols indicate double bonds where cleave. :carotenone C7–C8; : C9–C10; :C9′–C10′; C13–C14; :molecules C15–C15′; :means : C11′-C12′; : information C9′–C10′; ( ( , cleave. )) -dial ( Apo-10,10 ) Symbols indicateβ-carotene double bonds [19] where2 Crocetindial oxygenases : C7–C8; :When C9–C10; : C13–C14; : : C13′–C14′; : C11′-C12′; : suggested that one molecule of crocetindial and two of are released each d OH : C7′–C8′. an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold no product preference or No information Acycloretinal 2 × β-cyclocitral ( , ) -apocarotene-dial 3-OH-β-apo-13-carotenone × β-cyclocitral , Apo-10,10′-apocarotene zeaxanthinone ( ) 2 × hydroxi-βA -dial ( ) 3-OH-β-ionone ( ) Apo-13′Apo-13′-cantaOH n Astaxanthin ( ) Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 d No No No (in vitro) Apo-10,10′-apocarotene β-ionone ( ) + Apo-8,10′ β-apo-13γ-carotene cantaxanthinal ( ) ( )( :) Enzyme OH Lycopene Apo-13′cantaxanthinal ) double )4-oxo-β-inone References [19] [37] cleaves [38] [38] information Acycloretinal Zeaxanthin A 3-OH, 4-oxo-β-inone ) or information astax 3-OH, ( Retinal )[31] product astaxanthinone ) No No (in vitro) Nobond, 3-OH-β-ionone (cleaves ) cantaxanthinal ( ) ( positions. ( [36] ) () 3-OH-β-apo-13-carotenone d) (β-apo-14′-carotenal at theLack C15–C15′ equivalent Noof (bold No No (in vitro)(astaxanthinone No 2) [30,33] xOH 4-oxo-β-ionone ) RetinalOH Apo-12′When an -dial enzyme at different positions, the isNoindicated in bold. means no product preference Yes (in vivo) zeaxanthinal (No )or ( : ,C7′–C8′. 3-OH-β-ionone ( 3-OH-β-apo-14′-carotenal Apo-13′Astaxanthin β-ionone ( available. β-apo-13)) predominant Crocetindial ()cleave. , ) (the ((+ Apo-8,10′ )4-oxo-β-ionone cantax Apo-14′3-OH, 4-oxo-β-inone ( product xanthinone ( crocetindial ) β-apo-13-carotenone ( different β-carotene Symbols indicate double bonds whereatoxygenases C7–C8; C9–C10; :3-OH-retinal :)()(C15–C15′; : C13′–C14′; : indicated C9′–C10′; ( indicated )) C13–C14; :)not C7′–C8′. enzyme cleaves at positions, the is in bold. Lack of means no product preference or information No No poorly : C7′–C8′. When an enzyme cleaves different positions, is in( Apo-10,10′-apocarotene bold. Lack of bold no or information ,:-apocarotene-dial (β-apo-14′-carotenal -dial ) :When (product ) an3-OH-β-apo-13-carotenone carotenone (preference ) product molecule of [19]. OnNo the other hand, one molecule of and two molecules of ( ) ( β-carotene )predominant ( bold ) 2 Crocetindial ×cyclocitral hydroxi-β3-OH-β-ionone (β-apo-8′-carotenol ) ) No: C11′-C12′; Apo-13′3-OH-β-ionone )predominant Apo-13′Astaxanthin Astaxanthin Apo-13′0:-carotenal Apo-10,10′-apocarotene Apo-14′No means (in vitro) Echinenone ( 3-OH-β-apo-14′-carotenal Apo-10,10′-apocarotene OH double cantaxanthinal ( )zeaxanthinone 2Lutein ×bonds β-cyclocitral ) ( Apo-10,10′-apocarotene AAC9–C10; Unknown product OH Zeaxanthin Apo-12′Crocetindial () ( , , )oxygenases 3-OH, 4-oxo-β-inone (3-OH-β-apo-11-carotenal ) 3-OH-retinal astaxanthinone ( ) ( ) Symbols indicate where cleave. : C7–C8; : : C13–C14; C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; ? 3-OH-β-ionone ( ) β-apo-14 ( ) -apocarotene-dial ( ) carotenone ( ) OH n A not available. zeaxanthinal ( ) ( , 3-OH-β-ionone A ( ) Apo-10,10′-apocarotene Apo-14′Apo-14′Astaxanthin No No (in No vitro) No Retinal ) OHLack 3-OH-β-apo-14′-carotenal ( ( -dial )) 3-OH-retinal Apo-14′4-oxo-β-inone ( ) Nopreference ( ) 3-OH, 4-oxo-β-inone ( not astaxanthinone ( ) zeaxanthinone 3-OH-β-apo-13-carotenone 4-oxo-β-apo-8′-carotenal ( ) no ) zeaxanthin. 2 ×cyclocitral hydroxi-βd ( )is -dial ( ) astax : not C7′–C8′. When an Zeaxanthin enzyme cleaves at different positions, predominant indicated in( bold. of( bold means product information ) 3-OH, astaxanthinal ( astaxanthinone ) -dial ( Apo-10,10′-apocarotene )product Apo-12′available. Apo-13′Crocetindial ( , )the 3-OH-β-ionone ) ) available. Apo-13′d3-OH-β-apo-13-carotenone Astaxanthin hydroxyl-β-cyclocitral would beor released from a molecule zeaxanthinal (( )of Apo-10,10′-apocarotene Apo-14′No No No (in vitro) (bold. ) -dialLack Retinal Retinal 3-OH-β-ionone ( -dial )-dial β-apo-8′-carotenol β-apo-13-carotenone ( )) ?OH2.2.2. d the β-caroteneWhen an enzyme Apo-12′Crocetindial , ) No (Apo-10,10′-apocarotene 3-OH, astaxan ( ) ofSymmetrical astaxanthinal cantaxanthinal ( ) ( ) ( 3-OH-β-apo-14′-carotenal )3-OH-β-apo-11-carotenal : C7′–C8′. cleaves at positions, predominant product bold means no product preference or information β-apo-14′-carotenal CCDs with Mode of Action nin zeaxanthinal (( ))4-oxo-β-inone (hydroxi-β, ()4-oxo-β-inone Apo-10,10′-apocarotene Apo-14′-( )[31] Apo-130 Apo-10,10′-apocarotene Apo-14′3-OH-retinal ( is()(indicated ()(3-OH-α-apo-15′-carotenal )[30,33] Apo-14′cyclocitral References [19] [30,33] [36] [37] [38] cantaxanthinal ( ) 2 × β-cyclocitral ( ,different ) Apo-10,10′-apocarotene 3-OH-β-apo-13-carotenone ) References [19] [36] [31] [37] [38] [38] 4-oxo-retinal (slow) Not tested zeaxanthinone 2 × β-apo-13-carotenone 3-OH, ( ) astaxanthinone ( ) β-carotene d -dial ( ) astaxanthinal ( ) Lycopene -dial ( ) not available. 3-OH-β-apo-14′-carotenal ( ) No [30,33] No (in vitro) Yes (in Lutein zeaxanthinal ( ) Retinal Novivo) Zeaxanthin Apo-10,10′-apocarotene A Retinal ( [19] ) 3-OH-β-ionone ( β-ionone ) References [36]No [31] [37] [38] [38] (A3-OH-β-apo-11-carotenal ) 3-OH-retinal Retinal 2 Crocetindial × β-cyclocitral ( ,, , ()) )) Apo-10,10′-apocarotene Apo-10,10′-apocarotene Apo-13′Apo-12′Crocetindial ( ) astaxanthinal ( ) astaxanthinal ( ) zeaxanthinal ) ( Apo-10,10′-apocarotene , (()-dial No No poorly ) (? ) -dial ( ) [30,33] ( Astaxanthin ) -dial[31] 3-OH-β-apo-14′-carotenal not available. 3-OH-β-ionone )( [38] Apo-13′Apo-14′cyclocitral Apo-14′No No No (in vitro) No:(product Retinal ( (3-OH-retinal ) [36] References [19] indicate Unknown -dial (( )) bonds A 3-OH-β-apo-13-carotenone Symbols double where oxygenases cleave. : C7–C8; two :[38] : C13–C14; : C15–C15′; ( ): C13′–C14′; : C11′-C12′; : C Symbols double oxygenases :No C7–C8; : C9–C10; : [38] C15–C15′; C13′–C14′; C11′-C12′; : C9–C10; C9′–C10′; ( [37] ): C13–C14; zeaxanthinone ( ) A No -dial ( [37] ) : to astaxa Lycopene β-apo-14′-carotenal ( )cleave. Noindicate No (inbonds vitro) Apo-10,10′-apocarotene (? )3-OH-β-apo-13-carotenone Crocetindial -dial Yes vivo) Canthaxantin zeaxanthinone 2Int. × (hydroxi-βA17, 1781 dwhere β-carotene Microcystis, not identified yet, symmetrical zeaxanthinal References [36] [31] References [19] [30,33] [31] [37][30,33] [38] : from Apo-12′Crocetindial (()hydroxi-β,,Sci.))(2016, N N N carry out (3R)-3-OH-β-apo-8′-carotenol R 3-OH-β-apo-11-carotenal indicate bonds where cleave. : oxygenase C7–C8; C9–C10; : [38] C13–C14; : (in C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; A Mol. 9 of 37 [38] 3-OH, ( (((Apo-13′-canta) )) ) is able astaxanthinone ( ) 4-oxo-β-ionone )) ( [19] 2J.,× ((3-OH-α-apo-15′-carotenal -dial ) C Symbols astaxanthinal ( 4-oxo-β-inone ) zeaxanthinal Zeaxanthin Zeaxanthin No NoN 3-OH-β-ionone ( 3-OH-β-ionone )[36]double β-apo-14′-carotenal () )oxygenases N N N poorly N R 0:-carotenal 0 - means dd ( cleave. d3-OH-retinal Echinenone References [29] [32] -dial ) References [19] [30,33] [36] [31] [37] [38] 3-OH-β-apo-13-carotenone ( ) Apo-13′Unknown product 3-OH-β-apo-14′-carotenal ( ) Symbols indicate double bonds where oxygenases : C7–C8; : C9–C10; : C13–C14; C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; Apo-14′cyclocitral C d 3-OH-β-apo-14 ( ) Apo-14 2 x 4-oxo-β-ionone ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold no product preference or info an enzyme [36] cleaves atddifferent positions, the predominant product is[38] indicated in (in bold. Lack of bold means no preference or3-OH-retinal information Lycopene zeaxanthinal ) product Yes (in vivo) 0enzyme Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene Apo-12′Crocetindial (: C7′–C8′. ,[30,33] ) ( ,:When 3-OH-β-apo-11-carotenal ? cleavages ontheβ-carotene and at the and C7′–C8′ bonds [34]. has been [38] Nocleave. No No vitro) Apo-10,10′-apocarotene Apo-13′) no product Symbols indicate double bonds where : (3R)-3-OH-β-apo-8′-carotenol C7–C8; : zeaxanthin C9–C10; : No C13–C14; :3-OH-retinal C15–C15′; C13′–C14′; :No C11′-C12′; : C9′–C10′; J. Mol. Sci. 2016,where 17,[19] 1781oxygenases 9 of(:double 37 SymbolsReferences indicateInt.double bonds cleave. C7–C8; :When C9–C10; : 3-OH-retinal C13–C14; : C15–C15′; :[37] C13′–C14′; : C11′-C12′; C9′–C10′; :) C7′–C8′. an cleaves at[31] different positions, predominant product is:poorly indicated in C7–C8 bold. Lack of((xanthinone means preference or information -apocarotene-dial (inItvitro) No No 3-OH-β-apo-13-carotenone ((oxygenases No Apo-10,10 3-OH-β-apo-14′-carotenal )))) ( bold ) No 2 × cyclocitral zeaxanthinal (hydroxi-β, ) ( ) (is Apo-10,10′-apocarotene 3-OH-β-apo-13-carotenone Lycopene Unknown product double bonds wherezeaxanthinone oxygenases cleave. : C9–C10; : C13–C14; zeaxanthinal : C15–C15′; ( ): C13′–C14′; astaxanthinal : C11′-C12′; A: C7–C8; :)indicate Enzyme cleaves at the preference C15–C15′ double bond, or equivalent positions. 3-OH-retinal (of bold Zeaxanthin -dial (or(in ) information ( ) : C9 -dial ( ) product zeaxanthinal ( ( )) )Apo-14′:Lutein C7′–C8′. When an enzyme cleaves at different positions, in bold.Symbols Lack means no product Yes vivo) 3-OH-β-ionone not available. not available. 3-OH-β-apo-13-carotenone ( )indicated ) -dial ( A ) ( )the predominant zeaxanthinone 2 ×cyclocitral hydroxi-βA Apo-12′3-OH-retinal Npredominant N or information N in R)Lack No No poorly Symbols bonds where oxygenases C7–C8; :When : ((OH : that C13′–C14′; : C11′-C12′; : indicated C9′–C10′; suggested molecule of crocetindial and two molecules ofNβ-cyclocitral are released from each Apo-14′4-oxo-β-ionone )) C13–C14; :) C7′–C8′. cleaves different positions, theone product is bold. Lack of3-OH-retinal bold means no product preference or information : C7′–C8′.indicate When andouble enzyme cleaves at different positions, product is indicated in) bold. of bold means no product preference Crocetindial ,:not (( :3-OH-retinal ( at Zeaxanthin Apo-12′Crocetindial (cleave. , the ) d(predominant available. 3-OH-β-ionone ( C9–C10; ) d an 3-OH-β-apo-11-carotenal ?C15–C15′; -dial ( 3-OH-β-apo-14′-carotenal )enzyme 3-OH-β-apo-14′-carotenal ) Lutein 0 C d Echinenone β-ionone ( Unknown product References [19] [30,33] [36] [31] [37] [38] [38] No No No (in vitro) Apo-10,10′-apocarotene Lycopene 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-14′-carotenal ( ) 3-OH-β-apo-11-carotenal? Yes (in vivo) OH Apo-14′cyclocitral A cantaxanthinal ( predominant ) d, ) Apo-12 : C7′–C8′. When an enzyme cleaves at different positions, the product is indicated in bold. Lack of bold means no product preference or infor Z not available. cantaxanthinal ( ) OH zeaxanthinal ( ( Apo-10,10′-apocarotene 3-OH-retinal ( ) 3-OH-α-apo-15′-carotenal ) Apo-12′Z No No poorly No (in vitro) Apo-10,10′-apocarotene Apo-8′-lycopenol N N N N R molecule OH-dial ( )product is 3-OH-retinal (( )) Lack No[19]. different (positions, the indicated in bold. of bold means no product preference or information A of β-carotene On the other hand, one molecule and two Apo-13′molecules of not not: C7′–C8′. available.When an enzyme Nopredominant 3-OH-β-apo-14′-carotenal (OH Lutein cleaves at Canthaxantin Unknown product zeaxanthinal ( ) Aof crocetindial , () , Astaxanthin d available. d) OH ( )) Symbols 3-OH-retinal A 3-OH-β-ionone ( N) indicate N N NApo-12′double where : C7–C8; : Acycloretinal C13–C14; : C15–C15′; zeaxanthinal : C13′–C14′; ( ): C11′-C12′; : C9′–C10′; Crocetindial )C -dialA( 3-OH-β-apo-13-carotenone 3-OH-β-apo-11-carotenal ? 2.2.2. CCDsnot with Symmetrical Mode of Actioncleave. ( Apo-13′-canta): C9–C10; available. Lycopene -dial ( ) ( ) N bonds N oxygenases A Yes (incantaxanthinal vivo) No information 3-OH-α-apo-15′-carotenal Apo-8′-lycopenol OH 2 x3-OH-β-apo-13-carotenone 4-oxo-β-ionone ( ()3-OH, 3-OH-retinal ) Apo-12′Crocetindial ( , ) d No 3-OH-β-apo-14′-carotenal ( ) 3-OH-β-apo-11-carotenal ? not available. Lutein OH No poorly ( ) hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. 4-oxo-β-inone ( ) astaxanthinone ( ) d OH zeaxanthinal ( ) ( indicated ) (( )) Apo-13′- Axanthinone Astaxanthin (β-ionone ) Apo-10,10′-apocarotene cleaves at different positions, the predominant product is inAcycloretinal bold. Lack of bold means no product preference or information Unknown product A C 4-oxo-β-ionone dd 3-OH-β-ionone Z ( ) ( ) OH : C7′–C8′. When an enzyme OH Echinenone zeaxanthinal (No ) information 3-OH-α-apo-15′-carotenal A d 3-OH-retinal OH No 3-OH-β-apo-14′-carotenal ( ) Lycopene Apo-14′Lutein Canthaxantin C OH Apo-10,10′-apocarotene Lycopene Yes (in vivo) Apo-14′A β-carotene oxygenase from Microcystis, not identified to carry out two symmetrical 3-OH,A4-oxo-β-inone ( ) astaxanthinone Yes (in vivo) Ayet,( is) able OH Apo-10,10′-apocarotene d ) not available. N N 4-oxo-β-ionone ( ) OH3-OH-β-apo-13-carotenone -dial ( )( ) ( ) ( -dial Apo-13′-cantaNo No poorly NoN Nocantaxanthinal ( ) poorly ( ) Z Lycopene No 4-oxo-β-apo-8′-carotenal 3-OH-α-apo-15′-carotenal Yes (in vivo) ( ) astaxanthinal Echinenone 3-OH-retinal No OH Apo-10,10′-apocarotene Apo-14′2 x 4-oxo-β-ionone ( ) Unknown product OH -dial ( ) A Unknown product No No poorly L 3-OH-β-apo-14′-carotenal ( ) d Apo-10,10′-apocarotene cleavages on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ bonds [34]. It has been Y ) double xanthinone ( N N N A L Lutein 4-oxo-β-ionone ( ) Unknown product ( ) Y References [19] [30,33] [36] [31] [37] [38] [38] 3-OH-α-apo-15′-carotenal 4-oxo-retinal (slow) Not tested A C d No OH 4-oxo-β-apo-8′-carotenal N N astaxanthinal ( ) Echinenone β-ionone () -dial ) ( ) 3-OH-β-apo-13-carotenone ) N N -dial ( Apo-10,10′-apocarotene 3-OH-retinal ( ) ( OH w d No Apo-14′d Apo-10,10′-apocarotene d [38] 4-oxo-β-ionone ( )-dial ( 3-OH-β-apo-13-carotenone suggested that: one molecule of crocetindial andUtwowmolecules ofUAβ-cyclocitral are :released ( ))3-OH-β-apo-13-carotenone ( ) ) [19]C indicate [36] [31] (slow) Not from testedeach d β-ionone Canthaxantin OH NoSymbols Echinenone double[30,33] bonds 3-OH-β-apo-14′-carotenal where oxygenases : C7–C8; C9–C10; : [38] C15–C15′; : 4-oxo-retinal C13′–C14′; C9′–C10′; (cleave. ( [37] ): C13–C14; Lutein References 3-OH-α-apo-15′-carotenal cantaxanthinal ( : )C11′-C12′; -dial ( ( ) )( ) Apo-13′-cantaApo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal (OH) L Lutein OH Lutein 4-oxo-β-ionone Y 0 2 x 4-oxo-β-ionone ( ) molecule of :-carotenal β-carotene [19]. On in thebold. other hand, one molecule of crocetindial and two No : C7′–C8′. 3-OH-retinal (positions, ) OH3-OH-β-apo-14 References [29] [32] molecules of No is(indicated Echinenone Canthaxantin Symbols indicate double bonds where oxygenases cleave. : OH C7–C8; : C13–C14; C15–C15′; : C13′–C14′; :Lack C11′-C12′; : C9′–C10′; N N ) ( :) C9–C10; When an enzyme cleaves at different the predominant product of bold means no product preference or information β-ionone ( ) L xanthinone ( ) -dial ( ) Apo-10,10′-apocarotene 3-OH-retinal ( ) No U w Apo-13′-cantaL No L Apo-10,10′-apocarotene Y d 3-OH-α-apo-15′-carotenal 2 x4-oxo-β-ionone 4-oxo-β-ionone ) predominant References [29] [32] would be product released from axanthinone molecule zeaxanthin. Canthaxantin OHhydroxyl-β-cyclocitral Apo-14′β-ionone ( )( ( )the N atNthe preference :N cleaves C15–C15′ double or equivalent positions. 3-OH-retinal ) Enzyme : C7′–C8′. When an enzyme No cleaves at different positions, product is indicated in bold. Lack(ofNbold means no or information not available. -dial 3-OH-α-apo-15′-carotenal Echinenone (bond, )of U OH -dial (( )) w d Apo-13′-canta( OH ) Apo-10,10′-apocarotene 0 -carotenal( :) Enzyme cleaves at the cantaxanthinal OH α ( ) 2 x 4-oxo-β-ionone Canthaxantin β-ionone ( ) ( ) Apo-14′OH α ( OH C15–C15′ double bond, or equivalent positions. 3-OH-α-apo-15 ) No not available. L xanthinone ( ) Apo-13′-cantaOH 4-oxo-β-ionone -dial ( ) ( ) Apo-10,10′-apocarotene Echinenone 2 x4-oxo-β-ionone 4-oxo-β-ionone( ( ) ) Canthaxantin OH N Mode of Action cantaxanthinal Apo-14′-( ( ) ) xanthinone 2.2.2. CCDs with Symmetrical OH Apo-10,10′-apocarotene Echinenone L β-ionone Apo-13′-canta-dial ( () ) (4 )oxo β onone Apo-10,10′-apocarotene No 2 x 4-oxo-β-ionone Apo-10,10′-apocarotene E OH α cantaxanthinal Apo-14′-( ( ) ) -dial ( ) OH N Mode of Action E Echinenone xanthinone Canthaxantin No 2.2.2. CCDs with Symmetrical A apoca o ene d a -dial ((Apo )) Apo-10,10′-apocarotene -dial A 10 10 Apo-13′-cantaN ( ) yet, is able to carry out two symmetrical β-ionone ( ) ( ) A β-carotene oxygenase from Microcystis, cantaxanthinal not Apo-14′identified OH α N 2 x 4-oxo-β-ionone No d -dial β-ionone d ( () ) xanthinone ( ( ) ) Canthaxantin E cantaxanthinal Apo-10,10′-apocarotene A β-carotene from Microcystis, not identified yet, is able to carry out two symmetrical cleavages on β-caroteneoxygenase and zeaxanthin at the C7–C8 Apo-13′-cantaCanthaxantin Apo-14′-and C7′–C8′ double bonds [34]. It has been A 2 x 4-oxo-β-ionone ( ) -dial ( ) Apo-13′-cantaβ onone N 2 x 4-oxo-β-ionone xanthinone ( ( ) ) and E C cleavages onmolecule β-carotene and zeaxanthin at the C7–C8 bonds [34]. has been C ( d) suggested that one of crocetindial andcantaxanthinal two molecules of β-cyclocitral are released fromIteach AC7′–C8′ double Apo-10,10′-apocarotene A xanthinone A Apo-14′-( ) N Apo-10,10′-apocarotene -dial ( ) suggested that one[19]. molecule ofother crocetindial and two molecules of β-cyclocitral are released from d Apo-14′molecule of β-carotene On the hand, one molecule of crocetindial and two molecules of each A cantaxanthinal ( ) -dial ( ) A C A cantaxanthinal (A )molecule A d molecule of β-carotene [19]. On thefrom other one of crocetindial and two molecules of hydroxyl-β-cyclocitral would be released a hand, molecule of zeaxanthin. d C A of zeaxanthin. hydroxyl-β-cyclocitral would be released from a molecule A A Ad A d β-apo-13- Synechocystis carotenal ( sp. ) PCC 6803 -apocarotene-dial ) 3-OH-β-ionone ( )substrate + Apo- ( specificity 3-OH-β-apo-8′-carotenal β-apo-10′-carotenal Products Int. J. Mol. Sci. 2016, 3-OH-β-apo-10′-carotenal 17,β-apo-10′-carotenal 1781 10,10′-apocarotene TablePCC 2. Enzimatic activity and of different bacterial carotenoid oxygenases. Microcystis 7806 Nostoc sp.3-OH-β-ionone PCC 7120 7120 Novosphingobium alaskensis pacifica8 of 37 J. PCC Mol.and Sci. 2016, 17, 1781 ( Int. ) Enzymes Retinal ( 3-OH-β-apo-13-carotenone ) + Apo-tuberculosis

Int. J. Mol. Sci. 2016, 17, 1781

8 of 38

Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases.

13

39

Microcystis PCC 7806( ) + Nostoc sp. PCC 7120 PCC 7120 tuberculosis Novosphingobium alaskensiscarotenal ( ) pacifica ( minority , )( ) products -dial Transretinal ( ) β-ionone Apo-8,10′ β-apo-13β-apo-14′-carotenal carotenone ( ) Apo-10′Other Retinal ( ) β-apo-10′-carotenal 3-OH-β-ionone ) + ApoApo-12′β-ionone +(Apo-8,10′ ( ) β-apo-13-carotenone -apocarotene-dial ( )( )β-ionone carotenone β-apo-13-carotenone ( () ) ( ) β-apo-13-carotenone ( ) 3-OH-β-ionone + Apo)( +) )Apo-10,10′ β-ionone (( , 3-OH-β-apo-13-carotenone carotenal (( ) ) + Apo-10,10′ Transretinal 8,10′-apocarotene 3-OH-β-apo-13-carotenone β-apo-13-carotenone ( ) ( ( )) β-ionone(( )) + Apo-10,10′ β-apo-13-carotenone ( ) β-apo-13carotenal ( ) -apocarotene-dial 3-OH-retinal ( ) 10,10′-apocarotene -apocarotene-dial ( ) -apocarotene-dial ( ) -dial ( ) Retinal ( ) Retinal ( ) -apocarotene-dial ( ) Retinal 3-OH-retinal ( ) ( ) β-apo-13-carotenone ( ) ( β-apo-13-carotenone carotenone ( ) Apo-10′Other minority products )Retinal ( )β-apo-14′-carotenal β-ionone ( ) + Apo-10,10′ -dial ( ) Apo-10,10′-apocarotene 3-OH-β-ionone ) + ApoNo ( Transretinal No (in vitro) carotenal ( ) No ( , () -apocarotene-dial Retinal ( () ) ( No ) Retinal ) 3-OH-β-apo-13-carotenone ( ) -dial ( ) 3-OH-β-ionone ( ) + Apo3-OH-β-ionone ( ) +( 3-OH-β-apo-13-carotenone Apo3-OH-β-ionone ( ) + Apo3-OH-β-apo-8′-carotenal 10,10′-apocarotene ( ) β-apo-14′-carotenal ) β-ionone ( ) + Apo-8,10′ β-apo-13( ) 3-OH-β-apo-13-carotenone ( ) ( 3-OH-β-apo-13-carotenone )( ) 8,10′-apocarotene β-ionone-dial ( ) +( 3-OH-β-ionone Apo-10,10′ ( β-apo-13-carotenone )(3-OH-retinal + Apo) 8,10′-apocarotene 3-OH-retinal ( ) Apo-13′8,10′-apocarotene -apocarotene-dial ) carotenone ( ) 3-OH-β-apo-13-carotenone ( ) -dial ( ) -apocarotene-dial ( ) Retinal ( ) 3-OH-retinal ( ) 3-OH-retinal ( ) 8,10′-apocarotene 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( -dial ) 2 × hydroxi-βγ-carotene 3-OH-retinal ( ) -dial ( ) ( ) Zeaxanthin 3-OH-β-ionone ( ) 3-OH-β-apo-10′-carotenal ( () +)Apoβ-ionone ( 3-OH-β-ionone ) + 3-OH-β-apo-14′-carotenal Apo-8,10′ β-apo-13-dial β-carotene ( ) β-apo-13-carotenone (( )) Apo-14′3-OH-β-apo-13-carotenone 3-OH-β-ionone ( ) +( Apo3-OH-β-apo-8′-carotenal cyclocitral 2Apo-10,10′-apocarotene × β-cyclocitral ( , ) Apo-10,10′-apocarotene No No No (in vitro) 10,10′-apocarotene 3-OH-β-apo-10′-carotenal 3-OH-β-apo-10′-carotenal -apocarotene-dial ) carotenone ( ) 3-OH-β-apo-13-carotenone ( )( ) 3-OH-β-apo-10′-carotenal 3-OH-β-ionone +) Apo-3-OH-retinal 3-OH-β-ionone ( ) + ApoNo No No (in vitro) No Retinal 3-OH-β-ionone ( () +( )Apozeaxanthinal ( ) ( , ) 3-OH-retinal ( ) Crocetindial , ) -dial 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-13-carotenone ( ) -dial ( ( ) 8,10′-apocarotene -dial ( () ) 3-OH-retinal ( ) ( ) 10,10′-apocarotene Apo-12′Crocetindial ( , ) ? β-apo-14′-carotenal 10,10′-apocarotene 10,10′-apocarotene -dial ( )3-OH-β-apo-11-carotenal β-apo-13-carotenone ( ) β-carotene 3-OH-retinal ( ) 3-OH-retinal ( ) 3-OH-retinal ( ) γ-carotene 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene-dial ( ) Apo-13′zeaxanthinal ( ) ( ) Retinal ( ) -dial 3-OH-β-apo-10′-carotenal No No No( ) β-ionone ( -dial ) + Apo-8,10′ β-apo-13- No (in vitro) 3-OH-β-ionone ( ) + Apo3-OH-β-apo-13-carotenone ( ) ( ) 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( ) Lycopene γ-carotene Crocetindial ( , 2 ×) hydroxi-β- -dial -apocarotene-dial Yes (in vivo) Zeaxanthin (β-apo-14′-carotenal carotenone ( ) 3-OH-β-ionone ( )) ( ) No 10,10′-apocarotene No poorly β-ionone ( ) + Apo-8,10′ β-apo-13γ-carotene γ-carotene 3-OH-β-apo-14′-carotenal ( ) Apo-14′cyclocitral 3-OH-retinal ( ) Unknown product No No No (in vitro)Apo-13′Apo-10,10′-apocarotene -dial (β-ionone ) -apocarotene-dial ( ) + Apo-8,10′ β-ionone β-apo-13( ) + Apo-8,10′ β-apo-13( ) carotenone ( ) zeaxanthinal ( ) ( , ) (Int. ( ))J. Mol. Sci. 2016, 17, 1781 β-carotene 3-OH-β-apo-13-carotenone (β-apo-13-carotenone ) 3-OH-retinal -dial ( 3-OH-β-apo-13-carotenone ) ( ) zeaxanthinone ( ) 2 × hydroxi-βγ-carotene 2 × β-cyclocitral (( 3-OH-β-ionone , , )) Apo-10,10′-apocarotene Crocetindialβ-ionone Zeaxanthin -apocarotene-dial ( ) carotenone -apocarotene-dial ( ) (Apo-12′carotenone ( ) 3-OH-β-apo-11-carotenal ? ( ) No No No (in vitro) No) Retinal ( ) ( ) + Apo-8,10′ β-apo-133-OH-β-apo-14′-carotenal ( ) β-apo-13-carotenone ( ) Lutein β-carotene 3-OH-β-apo-14′-carotenal ( ) Apo-14′cyclocitral Crocetindial ( , ) -dial ( ) zeaxanthinal ( ) 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene No No ( ) No (in vitro) Apo-10,10′-apocarotene β-apo-14′-carotenal ( ) J.) Mol. -apocarotene-dial ( 3-OH-retinal ) carotenone (3-OH-retinal ) No 17, No No No (inzeaxanthinal vitro) No ( ) ( , ) ( ) Retinal (Int. Sci. 2016, 1781 ( , -dial ) ( ) -dial ( ) Yes (in vivo) β-apo-13-carotenone ( ) β-apo-13-carotenone ( ) β-carotene Lycopene Crocetindial ( Crocetindial β-carotene can 3-OH-α-apo-15′-carotenal , ) β-apo-14′-carotenal ( ) 3-OH-β-apo-11-carotenal ? No 2 × No ( , poorly Apo-12′- Apo-13′Enzymes and Products 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene β-cyclocitral ) Apo-10,10′-apocarotene β-apo-13-carotenone ( ) β-carotene Unknown 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( (in ) vitro) Retinal ( 2 × hydroxi-β3-OH-β-ionone ) product No No No No No N zeaxanthinal ( ) (Apo-13′2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene Retinal ( ) Astaxanthin ) No ( ( )) Zeaxanthin 3-OH-β-ionone No No No (in )vitro) No( ) Apo-14′Retinal ( Mol. ) Sci. 2016, 17,( 1781 Int. J. 9 of 37asta Crocetindial ( , ) -dial ( ) Crocetindial ( , -dial Assayed Substrates 3-OH-β-apo-14′-carotenal ) 3-OH-β-apo-13-carotenone ( ) 3-OH, 4-oxo-β-inone ( ) 3-OH-β-apo-13-carotenone ( ) β-carotene-oxygenase NosCCD (NSC1) Nostoc MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis Crocetindial ( 4-oxo-β-ionone , cyclocitral ) -dial ( ) Lycopene 2 × hydroxi-β( ) No No (in vitro)Yes (in vivo)zeaxanthinone ( )β-apo-14′-carotenal ( ) Apo-10,10′-apocarotene ( No) canta Zeaxanthin Echinenone 3-OH-β-ionone ( ) β-apo-14′-carotenal ( β-apo-14′-carotenal ) No poorly zeaxanthinal ( ) ( cyclocitral , ) 3-OH-β-apo-14′-carotenal (No )) 3-OH-retinal ( ) Lutein Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal ( Apo-14′Apo-10,10′-apocarotene Unknown product -dial7120 ( ) Microcystis PCCCrocetindial 7806 ( , ) sp. Apo-10,10′-apocarotene PCC tuberculosis Astaxanthin PCCNo7120 alaskensis pacifica 3-OH-β-ionone ( ) No NoNovosphingobium (in vitro) No Apo-13′Apo-13′Apo-12′3-OH-β-apo-11-carotenal 3-OH-retinal ( ( ) )? No -dial ( zeaxanthinal ) ast Apo-12′( ) ( ,( )) -dial 3-OH-retinal (( )) 3-OH, 4-oxo-β-inone astax -dial 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-13-carotenone zeaxanthinone ( ) ( ) ( )3-OH-β-apo-13-carotenone 2 × hydroxi-βzeaxanthinal Int. J. Mol. 17, 03-OH-β-apo-13-carotenone ( )1781 2 × hydroxi-β- [19] ( ) ( [31]Apo-13 3-OH-α-apo-15′-carotenal ) 2 × hydroxi-βReferences [30,33] [36] zeaxanthinone [37] [38] cantaxanthinal ( ) Apo-12′Crocetindial ( ), ) Zeaxanthin β-ionone (3-OH-β-ionone 3-OH-β-apo-11-carotenal ? Sci. 2016, ( ) 3-OH-β-apo-14′-carotenal ( )) Lutein Zeaxanthin Zeaxanthin Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal ( 3-OH-β-ionone ( ) 3-OH-β-ionone ( ) Apo-14′cyclocitral Lycopene Yes (in vivo) (3-OH-β-apo-14′-carotenal (Astaxanthin ) zeaxanthinal ) 3-OH-β-ionone ( ) Apo-13′No No No (in vitro) Apo-10,10′-apocarotene Canthaxantin 3-OH-β-apo-14′-carotenal ( ) ( ) Canthaxantin Apo-14′cyclocitral cyclocitral Symbols No indicateNodoubleNo bonds where oxygenases cleave. : No C7–C8; C9–C10; : C13–C14; : No poorly zeaxanthinal 3-OH-retinal (( )) -dial (( ) ) astax ( , ) J.: Mol. Sci. 2016, 17,canta-xanthinone 1781 : C15–C15′; Apo-13′-cantaNo (inInt. vitro) No ( ): C13′–C14′; astaxanthinone No: C11′-C12′; N Apo-10,10′-apocarotene Apo-10,10′-apocarotene Unknown product 4-oxo-β-inone ( ) ( ) )) 2 (x-dial 4-oxo-β-ionone (( 3-OH-retinal Lycopene (in vivo) ) ( )4-oxo-β-ionone Int. J. Mol. Sci.( 2016, 1781 Echinenone 3-OH-α-apo-15′-carotenal zeaxanthinal ([31])Apo-14 ,) ) ( , No ) 3-OH, References [19]atxanthinone [30,33] Yes [36] is indicated [37] ) 17, 3-OH-retinal ( ) in bold. Crocetindial 2( (x ,4-oxo-β-ionone ? 3-OH-retinal No poorly ( positions, ) Apo-12′different predominant product Lack of 0bold means no product[38] preference orcanta inf 0 -apocarotene-dial Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene 3-OH-β-apo-13-carotenone ( ) : C7′–C8′. When an enzyme cleaves -dial (3-OH-β-apo-11-carotenal ) -dial ( )(the Unknown product Apo-10,10′-apocarotene Int. Sci. 17, 1781 9 of 37 Int. J. 2016, Mol. Sci. 17, 1781 9 of 37 Apo-10,10 Int.J.Int. J.Mol. Mol. Sci. 2016, 17,2016, 1781 9 of 37 J. Mol. Sci. 2016, 17, 1781 9 of 37 No ( ) zeaxanthinal ) Apo-14′Apo-12′Crocetindial ( , ) Crocetindial ( where , ) -dial 3-OH-β-apo-11-carotenal ? 3-OH-β-apo-11-carotenal ?cantaxanthinal indicate double bonds oxygenases cleave. (: C7–C8; : C9–C10; : C13–C14; : C15–C15′; ( ): C13′–C14′; astaxanthinal : C11′-C12′; ( ) ( ) : C 3-OH-β-apo-14′-carotenal ( Symbols )not Lutein ) -dial ( )4-oxo-β-ionone ( )-dial ( ) Astaxanthin Int. J. Mol. Sci. 2016, 17, 1781 9 3-OH-β-ionone of 37 3-OH-β-apo-13-carotenone ( ) available. ( β-ionone ) Lycopene cantaxanthinal ( Yes ) (in vivo) A 0 - means Echinenone ( ) zeaxanthinal ( [37] )Lack References [19] [30,33] [36] [38] no product preference [38] 3-OH, 4-oxo-β-inone ( ) astax 3-OH-retinal ( ) Nocleaves No No poorly Apo-12 :( C7′–C8′. When an enzyme at different positions, the predominant product is [31] indicated in bold. of bold orcantax info 3-OH-β-apo-14′-carotenal ) Lutein Apo-10,10′-apocarotene Unknown product No Apo-10,10′-apocarotene 3-OH-α-apo-15′-carotenal Lycopene Canthaxantin Lycopene Apo-12′Apo-12′Symbols indicate bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; 3-OH-retinal ( )notdouble Apo-12′No Apo-12′Yes (in vivo) cantaxanthinal ( ) -dial ( ) Apo-12′- 3-OH-β-iononeApo-13′-cantaavailable. ( ) A Astaxanthin 3-OH-β-apo-13-carotenone 2 x 4-oxo-β-ionone ( ) -dial ( ) asta No No poorly No No ( () ) 3-OH-β-io 3-OH-α-apo-15′-carotenal β-ionone ( ) cantaxanthinal ( ) 4-oxo-β-inone xanthinone ) ( (bold. ) ) (Astaxanthin cantaxanthinal 3-OH, ( cantaxanthinal ) cantaxanthinal astaxa cantaxanthinal ) ( )of bold Unknown product : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is( indicated in Lack means no product[37] preference or information 3-OH-β-apo-14′-carotenal ( ) Lutein Apo-10,10′-apocarotene References [19] [30,33] [36] [31] Apo-130 [38] 4-oxo-β-ionone 3-OH-β-ionone (( )) ( ) CanthaxantinEchinenone Apo-14′3-OH, 4-oxo 3-OH-β-ionone ( ) Apo-13′Astaxanthin Apo-10,10′-apocarotene A Astaxanthin 3-OH-β-ionone ( ( ) ) ( ) -dial Apo-13′( 3-OH-β-apo-13-carotenone )available. 3-OH-β-ionone ( ) ( ) 3-OH-retinalnot Apo-13′Astaxanthin Astaxanthin 3-OH-β-ionone Apo-13′3-OH-β-ionone Apo-13′Astaxanthin Astaxanthin Apo-13′-canta( ) No ( astaxanthinone ) : C15–C15′; ( ): C13′–C14′; : C11′-C12′; astaxa (3-OH-β-apo-13-carotenone ) : C9–C10; 4-oxo-β-ionone Symbols indicate double bonds where oxygenases : C7–C8; : C13–C14; : C 3-OH, 4-oxo-β-inone ( ) Apo-10,10′-apocarotene astaxanthinone ( ) cleave. 4-oxo-β-inone (( )) 4-oxo-β-ionone ( ) -dial ( cantaxanthinal ) No 2 x3-OH, Apo-10,10′Echinenone xanthinone ( ) 3-OH, 4-oxo-β-inone ( (0 Apo-10,10′-apocarotene ) ) -dial astaxanthinone ( ( ) )( ) ( ) ( ) 3-OH, 4-oxo-β-inone astaxanthinone 3-OH, 4-oxo-β-inone astaxanthinone 3-OH, 4-oxo-β-inone ( ) ( ( ) ) 3-OH-α-apo-15′-carotenal astaxanthinone 0 3-OH-β-apo-14′-carotenal ( ) 3-OH-β-apo-14′-carotenal Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene Lutein Lutein References [19] [30,33] [36] [31] [37] [38] Apo-10,10 -apocarotene-dial Apo-14 - means no product preference or-dia : C7′–C8′. When an enzyme cleaves at different positions, of bold info No ( ) Apo-14′-the predominant product is indicated in bold. Lack Apo-10,10′-apocarotene Apo-14′- ( ) -dial( ( ) ) Apo-10,10′-apocarotene Apo-14′-dial Apo-10,10′-apocarotene ( ) astaxanthinal ( ) Apo-10,10′-apocarotene Apo-14′-dial ( ) β-ionone 3-OH-retinal ( indicate ) 3-OH-retinal No cleave. ( ): C7–C8;Apo-14′No cantaxanthinal 4-oxo-β-ionone (( )) Symbols double bonds where oxygenases : C9–C10; : C13–C14; astaxanthinal : C15–C15′; ( ): C13′–C14′; : C11′-C12′; : [30 C9 not available. References [19] ( ) References EchinenoneCanthaxantin[19] [30,33] [36] [31] [37] [38] [38] -dial (-dial astaxanthinal ( ) astaxanthinal ( ) -dial ( ) )-dial astaxanthinal ( ) ( ) ( ) β-ionone astaxanthinal ( ) Apo-13′-cantaApo-10,10′-apocarotene 3-OH-α-apo-15′-carotenal 3-OH-α-apo-15′-carotenal 2 x 4-oxo-β-ionone ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product preference or infor Canthaxantin No xanthinone ( ) [38] References [30,33] [36] [31] [37] [38] [38] Symbols indicate double bonds where oxygenases cleave. : -dial C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; ( ) References [19] [30,33] [36] [31] [37] [38] References [30,33] [36] [31] [37] [38] [38] Apo-13′-cantaReferences [19] [30,33] [36] [31] [37] [38] [38] References [19] [19] [30,33] [36] [31] [37] [38] [38] Apo-10,10′-apocarotene (not )available. ( ) 2 x 4-oxo-β-ionone ( ) Apo-14′β-ionone ( ) -dial ( ) ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product0 preference 0or information 0 4-oxo-β-ionone 0 cantaxanthinal 0 (xanthinone 0 0 0 Apo-10,10′-apocarotene (0 ) 4-oxo-β-ionone ( ) ) Canthaxantin Apo-14′Echinenone Echinenone -dial ( ) Apo-13′-cantanot available. cantaxanthinal ( ) 2 x 4-oxo-β-ionone ( ) Apo-10,10′-apocarotene Apo-10,10′-apocarotene xanthinone ( ) No No Apo-10,10′-apocarotene-dial ( ) -dial ( ) Apo-14′-dial ( ) cantaxanthinal β-ionone ( ) β-ionone ( ( ) )

β-apo-10′-carotenal 3-OH-β-apo-8′-carotenal

3-OH-β-apo-10′-carotenal β-apo-10′-carotenal β-apo-8′-carotenal β-apo-10′-carotenal β-carotene 2 × β-cyclocitral ( , ) 3-OH-β-apo-10′-carotenal Crocetindial ( , ) γ-carotene 3-OH-β-apo-8′-carotenal 3-OH-β-apo-8′-carotenal β-apo-10′-carotenal 3-OH-β-apo-8′-carotenal

(3R)-3-OH-β-apo-12′-carotenal

3-OH-retinal

3R-3-OH-retinal

Acycloretinal (slow)

Acycloretinal

No information

Acycloretinal

Retinal

Retinal

3-OH-retinal

3-OH-retinal

No information

Acycloretinal

4-oxo-retinal (slow)

Not tested

Apo-8′-lycopenal

Int. J. Mol. Sci. 2016, 17, 1781

9 of 38

Apo-10′-lycopenal

Table 2. Cont.

β-apo-8′-carotenol

(3R)-3-OH-β-apo-8′-carotenol

Apo-8′-lycopenol

4-oxo-β-apo-8′-carotenal

References [29] Symbols indicate [32] double bonds where oxygenases cleav Symbols indicate double bonds where oxygenases cleave. : :C7–C8; : :C9–C10; : :C13–C14; : :C15–C15′; C11′-C12′; : :C9′–C10′; Symbols indicate double bonds where oxygenases cleave. C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; :; C9′–C10′; Symbols indicate double bonds where oxygenases cleave. C7–C8; C9–C10; C13–C14; C13′–C14′; C11′-C12′; C9′–C10′; Symbols indicate double bonds where oxygenases cleave. : C7–C8; : :C9–C10; C13–C14; : C15–C15′; :atC13′–C14′; : double C11′-C12′; : C9′–C10′; : C7′–C8′. :C15–C15′; Enzyme cleaves the C15–C15′ or equivalent positions. Symbols indicate double bonds where oxygenases cleave. : C7–C8; C9–C10; : C13–C14; C15–C15 ; : :C13′–C14′; C13 –C14 ; : :C11 -C12 ; bond, : C9 –C10 C7 –C8 . When When an an enzyme enzyme cleaves at different positions, cleaves at different positions, product is indicated inproduct bold. Lack ofindicated bold means noLack product preference or information notor available. : C7′–C8′. When an enzyme cleaves at positions, the product isisindicated ininbold. Lack of means no preference : C7′–C8′. When an enzyme cleaves atpredominant different positions, the predominant product is in bold. Lack of bold means no product preference or information : C7′–C8′. When an enzyme cleaves atdifferent different positions, thepredominant predominant product indicated bold. Lack ofbold bold means noproduct product preference orinformation information : C7′–C8′. When an enzyme cleaves atthe different positions, the predominant is indicated in bold. of bold means no product preference or information not available. not 2.2.2. CCDs with Symmetrical Mode of Action not available. notavailable. available. not available. Canthaxantin

Canthaxantin

2 x 4-oxo-β-ionone ( ) Apo-10,10′-apocarotene -dial ( )

Apo-13′-cantaA β-carotene oxygenase from Microcystis, not identified yet, is able to carry out two symmetrical 2 x 4-oxo-β-ionone ( ) xanthinone ( ) Apo-10,10′-apocarotene cleavages on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ double bonds [34]. It has been Apo-14′) suggested that one molecule of crocetindial and -dial two( molecules of β-cyclocitral are released from each cantaxanthinal ( ) molecule of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin.

Retinalsubstrates. ( ) showing products to different

β-apo-10′-carotenal

( , ) β-ionone ( ) + Apo-10,10′

Int. J. Mol. Sci. 2016, 17, 1781 Int. J. Mol. Sci. 2016, 17, 1781

3-OH-β-apo-8′-carotenal 3-OH-β-apo-8′-carotenal

Table 3. Substrates tested and cleaved with a unique 3-OH-β-apo-10′-carotenal Table 3. Substrates tested and cleaved with a unique Substrates 3-OH-β-apo-10′-carotenal Oxygenase Substrates Oxygenase β-apo-4′-carotenal β-apo-4′-carotenal γ-carotene Int. J. Mol. Sci. 2016, 17, 1781

Int. J. Mol. Sci. 2016, 17, 1781

Int. J. J. Mol. Mol. Sci. Sci. 2016, 2016, 17, 17, 1781 1781 Int.

γ-carotene

NACOX1 NACOX1

Transretinal ( )

β-apo-13-carotenone ( Apo-8′-lycopenal

β-apo-10′-carotenal

)

Diox1

( ) Retinal ( ) ( ) β-ionone ( ) + -apocarotene-dial Apo-10,10′ 10 of 38 β-apo-13-carotenone Substrates Acycloretinal (s Synechocystis sp. PCC 6803 -apocarotene-dial 10 ( )of 38 Retinal ( ) 3-OH-β-ionone ( ) + Apo3-OH-β-apo-13-carotenone ( ) β-apo-4′-carotenal 8,10′-apocarotene 3-OH-β-ionone ( ) + Apo- Apo-10′-lycopenal 3-OH-retinal 3-OH-β-apo-13-carotenone ( ) ( ) Retinal 8,10′-apocarotene-dial ( ) oxygenase. No informatio 3-OH-retinal ( ) oxygenase. ( ) + Apo-dial3-OH-β-ionone ( ) 3-OH-β-apo-13-carotenone ( ) Products 3-OH-β-ionone ( 10,10′-apocarotene )β-apo-8′-carotenal + Apo3-OH-retinal ( ) 3-OH-β-apo-13-carotenone Products 10 (of) 38 ) Retinal 10,10′-apocarotene-dial ( β-apo-8′-carotenol 3-OH-retinal ( ) -dial ( ) Retinal β-ionone (of 38 ) + Apo-8,10′ 10 10 of 38 β-apo-13-carotenone ( ) β-apo-10′-carotenal ( ) β-apo-13-carotenone β-ionone (( )) +-apocarotene-dial Apo-8,10′ β-apo-13-

Retinal carotenone -apocarotene-dial ( (3R)-3-OH-β-apo-8′-carotenol ) β-apo-13-carotenone ( ) β-carotene 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene 3-OH-retina Int. J. Mol. Sci. 2016, 17, 1781 No 3,3′-dihydroxy-isorenieratene Retinal ( ) 3-OH-β-ionone ( ) Table Astaxanthin 3. Substrates Substrates tested tested and cleaved cleaved with with aa unique unique oxygenase. β-carotene Int. J. Mol. Sci. 2016, 17,3,3′-dihydroxy-isorenieratene 1781 10-dial of 37 Crocetindial ( , ) ( ) β-apo-13-carotenone ( ) Table 3. and oxygenase. β-apo-12′-carotenal 2 × β-cyclocitral ( , ) ( Apo-10,10′-apocarotene ( ) No Int. J. Mol. Sci. 2016, 17, 1781 10 of 37 3-OH, 4-oxo-β-inone ) No Retinal β-apo-14′-carotenal ( ) 3-OH-β-apo-13-carotenone ( ) Crocetindial ( , ) -dial ( ) Retinal 9 of 37 Substrates Oxygenase Products Mol.17, Sci.1781 2016, 17, 1781 Substrates Apo-10,10′-apocarotene Oxygenase Int. J. Mol. Int. Sci. J. 2016, 3-OH-β-apo-13-carotenone ( ) 10 of 37 Apo-8′-lycopenol β-apo-14′-carotenal ( ) Substrates Oxygenase Products Products MtCCO 3-OH-β-apo-15′-carotenal ( ) β-apo-4′-carotenal 3-OH-β-apo-13-carotenone ( ) -dial 2( × hydroxi-β) MtCCO No informatio 3-OH-β-apo-15′-carotenal ( ) β-apo-4′-carotenal Zeaxanthin 0 -carotenal 3-OH-β-apo-14′-carotenal ( )3-OH-β-ionone ( ) 3-OH-β-apo-14′-carotenal ( [37] ) cyclocitral References [19] with a unique [30,33] [36]of 373-OH-β-apo-13-carotenone [31] [38] 3-OH-β-ionone ( ) Astaxanthin tested and cleaved Table 3. Substrates oxygenase. Int. J. Mol. Sci. 2016, 17, β-apo-4 1781 10 (3R)-3-OH-β-apo-8′-carotenal ( ) 3-OH-β-apo-14′-carotenal ( Apo-10,10′-apocarotene ) 2 × hydroxi-βNo Apo-12′Zeaxanthin ( ) Table 3. Substrates tested and cleaved NACOX1 with a unique oxygenase. ( , ) 3-OH-β-ionone 3-OH, 4-oxo-β-inone ( ) ( ) 3-OH-retinal β-apo-13-carotenone 3-OH-β-apo-14′-carotenal ( ) ( ) Int. J. Mol. Sci. 2016, 17, 1781 10-dial of4-oxo-β-apo-8′-carotenal 37 cyclocitral Symbols indicate double bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; NACOX1 ) β-apo-13-carotenone ( NACOX1 ( ) 3-OH-retinal β-apo-13-carotenone ( ) cantaxanthinal ( : No )C11 No Apo-10,10′-apocarotene Table 3. Substrates tested and cleavedOxygenase with a unique oxygenase. Substrates Products Crocetindial ( , ) Apo-10,10′-apocarotene 3-OH-β-apo-11-carotenal ? Echinenone ( , ) 3-OH-retinal ( ) 3-OH-β-ionone ) different positions, Apo-13′Astaxanthin SubstratesWhen an enzyme Products -dial (product Echinenone ) 4-oxo-retinal (sl : C7′–C8′. cleaves( atOxygenase the predominant is indicated in bold. Lack of bold means no product prefe β-apo-4′-carotenal ) Crocetindial ( ,-dial ) (Products 3-OH-β-apo-11-carotenal ? 3-OH, 4-oxo-β-inone Oxygenase ( ) astaxanthinone ( ) Substrates β-apo-4′-carotenal Table 3. Substrates with Lycopene References tested and cleaved [19] a unique oxygenase. [30,33] [36] [31] [37] [ NACOX1 β-apo-13-carotenone ( (3R)-3-OH-β-apo-12′-carotenal ) ) not available. Apo-10,10′-apocarotenoid ( 3,3′-dihydroxy-isorenieratene 0 Apo-10,10′-apocarotene Apo-14′No β-apo-4′-carotenal 3,3′-dihydroxy-isorenieratene NosCCD 3,3 -dihydroxy-isorenieratene NACOX1 β-apo-13-carotenone ( () ) Apo-10,10′-apocarotenoid Table 3. Substrates tested and cleaved with aoxygenases unique oxygenase. 2 × C13 :( C7–C8; ) [29] Lycopene NosCCD Symbols bonds where cleave. : C9–C10; (References : C15–C15′; 3-OH-retinal : C13′–C14′; ): C13–C14; 3-OH-β-apo-13-carotenone -dial ( ) astaxanthinal ( ) : C Substrates indicate double Oxygenase NACOX1 3-OH-β-apo-13-carotenone β-apo-13-carotenone ( )(( )) 2Products × C13 ( ) No No 3-OH-β-apo-13-carotenone 3,3′-dihydroxy-isorenieratene 3-OH-β-apo-13-carotenone ( ) 0 References [19] [30,33]cleaves [36] [31] [37] [38] [38]product MtCCO :) Enzyme at the C15–C15′ double o 3-OH-β-apo-15 Int. J. Mol. Sci. 2016, 17, 1781 9bond, of 37 p : C7′–C8′. When an enzyme at different positions, the predominant product is(indicated in cleaves bold. Lack of bold means no β-apo-4′-carotenal MtCCO 3-OH-β-apo-13-carotenone Substrates Oxygenase Products 3-OH-β-apo-15′-carotenal ( ( )-carotenal 3,3′-dihydroxy-isorenieratene MtCCO 3-OH-β-apo-14′-carotenal ( ) 3-OH-β-apo-15′-carotenal ( )) Lutein Int. J. 3-OH-β-apo-13-carotenone Mol. Sci. 2016, 17, 1781 3-OH-β-apo-13-carotenone ) MtCCO β-apo-4′-carotenal 3-OH-β-apo-14′-carotenal 3-OH-β-apo-15′-carotenal Symbols indicate double bonds oxygenases cleave. : C7–C8; : C9–C10; : C13–C14;( ((()( 0):))-carotenal C15–C15′; : ( C11′-C12′; Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 : C9′–C10′; NACOX1 β-apo-13-carotenone 3-OH-β-apo-14 ( ): C13′–C14′; 3,3′-dihydroxy-isorenieratene Apo-8′-lycopenal notwhere available. 3-OH-β-apo-14′-carotenal 3-OH-retinal ( ) Nostoxanthin 3-OH-β-apo-14′-carotenal ( ) 3-OH-β-apo-13-carotenone MtCCO Lutein 3-OH-β-apo-15′-carotenal ( ( )) 3-OH-β-apo-14′-carotenal Nostoxanthin NACOX1 β-apo-13-carotenone ( () Lack Apo-12′-(slow) Acycloretinal : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in 2.2.2. bold. of bold means no3-OH-α-apo-15′-carotenal product or information of Action MtCCO 3-OH-retinal ( Mode ) preference No 3-OH-β-apo-15′-carotenal (( ))CCDs with Symmetrical 3-OH-β-apo-14′-carotenal cantaxanthinal ( ) 3,3′-dihydroxy-isorenieratene Echinenone Apo-14′-nostoxanthinal ( ( ( ) )) Echinenone Echinenone ( Apo-12′) 15 5 3-OH-α-apo-15′-carotenal 3-OH-β-apo-14′-carotenal not available. 3-OH-β-apo-13-carotenone Echinenone Apo-14′-nostoxanthinal ( )A β-carotene oxygenase from 3-OH-β-ionone ( ) PpCCO Apo-13′Astaxanthin 0 Echinenone 3,3′-dihydroxy-isorenieratene cantaxanthinal ( ) not Microcystis, identified ye Apo-12′-nostoxanthinal (( ) )4-oxo-β-ionone (( )) PpCCO Apo-10,10 -apocarotenoid Apo-10,10′-apocarotenoid MtCCO ( ) Apo-10′-lycopenal 3-OH-β-apo-13-carotenone ( 3-OH-β-apo-15′-carotenal ( ) Echinenone Apo-12′-nostoxanthinal ( )) 4-oxo-β-inone ( NosCCD ) NosCCD astaxanthinone ( ) Echinenone Apo-10,10′-apocarotenoid 3-OH-β-ionone ( 3-OH, ) Apo-13′Astaxanthin Apo-10,10′-apocarotenoid 2 × C1340(4-oxo-β-ionone ) 2cleavages theinformation C7–C8 3-OH-β-apo-14′-carotenal ))) (( ))on β-carotene and zeaxanthin at No × (((((CApo-10,10′-apocarotene MtCCO NosCCD 3-OH-β-apo-15′-carotenal Apo-10,10′-apocarotenoid 13 NosCCD Apo-14′- and C7 Apo-10,10′-apocarotenoid ) 22No ×× C 13 )) 3-OH, 4-oxo-β-inone Apo-10,10′-apocarotene ( Echinenone ) astaxanthinone ( ) NosCCD -dial ( ) C 13 ( ( NosCCD ( ) 2 × CApo-10,10′-apocarotene 13 ( ) -dial ( ) astaxanthinal ( ) that one molecule of crocetindial and two molecules o No3-OH-β-apo-14′-carotenal 2 × C13 ( ) suggested Apo-10,10′-apocarotene Apo-14′Echinenone Hydroxylycopene -dial ( ) β-ionone ( ) Nostoxanthin Hydroxylycopene References [19] -dial ( ) [30,33] Canthaxantin [36] [31] [37] [38] [38] astaxanthinal ( hand, ) Echinenone Apo-10,10′-apocarotenoid ( ) () )β-apo-8′-carotenol Nostoxanthin Nostoxanthin molecule of β-carotene [19]. On the other one molecule β-ionone Apo-14′-nostoxanthinal ( NosCCD 0 PpCCO References Symbols indicate double [19] [30,33] [36] [31] [37] [38] [38] 2Unknown × C13 ( )-nostoxanthinal Nostoxanthin Apo-14′-nostoxanthinal (( ( :)2) )xC15–C15′; bonds where oxygenases cleave. : C7–C8; : C9–C10; :Apo-14 C13–C14; C11′-C12′; C9′–C10′; ( (): C13′–C14′; Apo-10,10′-apocarotenoid 4-oxo-β-ionone ) Nostoxanthin PpCCO Apo-12′-nostoxanthinal Canthaxantin PpCCO hydroxyl-β-cyclocitral would be: released from: aRetinal molecule of ze NosCCD Nostoxanthin PpCCO PpCCO 0)-nostoxanthinal ( :Apo-10,10′-apocarotene Apo-12′-nostoxanthinal ) C13′–C14′; 2Unknown × C132 (xin Symbols indicate: C7′–C8′. double bonds where oxygenases C7–C8; C9–C10; : Apo-12 C15–C15′; C9′–C10′; or information ): C11′-C12′; 4-oxo-β-ionone (of)bold(means When an enzyme cleaves atcleave. different the: 17, predominant productApo-14′-nostoxanthinal is indicated bold. Lack no product: preference PpCCO : C13–C14; Int. J.:positions, Mol. Sci. 2016, 1781 Apo-12′-nostoxanthinal ( ) -dial ( ) Nostoxanthin Apo-14′-nostoxanthinal ( ) Hydroxylycopene Apo-10,10′-apocarotene Apo-14′-nostoxanthinal ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product preference or information PpCCO (3R)-3-OH-β-apo-8′-carotenol not available. Int. J. Mol. Sci. 2016,PpCCO 17, 1781 Hydroxylycopene Apo-14′-nostoxanthinal (( )) Apo-12′-nostoxanthinal Nostoxanthin Apo-12′-nostoxanthinal ( () ) Dihydroxylycopene PpCCO Unknown -dial Hydroxylycopene 3-OH-retinal not available. Hydroxylycopene Apo-12′-nostoxanthinal Dihydroxylycopene Table 2. Unknown Enzimatic activity and substrate specificity of different bacterial carotenoid oxy Apo-14′-nostoxanthinal ( ) PpCCO PpCCO Apo-12′-nostoxanthinal ( ) activity and substrate specificity of different bacterial caroteno PpCCO PpCCO Unknown Unknown Table 2. Enzimatic Dihydroxylycopene PpCCO Unknown Hydroxylycopene Hydroxylycopene Apo-8′-lycopenol Hydroxylycopene Enzymes and Products PpCCO Unknown Dihydroxylycopene Sci. 2016, 17, 1781 Hydroxylycopene Int. J. Mol.Assayed PpCCO No and information Table Unknown 1. Enzymatic activity of MtCCO bacterial apo-carotenoid cleavage oxy Substrates β-carotene-oxygenase NosCCD (NSC1) Mycobacterium NSC3 Nostoc sp. NA Dihydroxylycopene Enzymes Products PpCCO Unknown Int. J. Mol. Sci. 2016, 17, 1781 Dihydroxylycopene PpCCO Unknown PpCCO Unknown Microcystis PCCUnknown 7806 Nostoc sp.NosCCD PCC 7120 7120 Nostoc Novosp tuberculosis Assayed Substrates PpCCO β-carotene-oxygenase (NSC1)substrates. MtCCO Mycobacterium PCCNSC3 sp. PpCCO Unknown showing products to different Int. J.Dihydroxylycopene Mol.Torulene Sci. 2016, 17, 1781 Table 2. Enzimatic activity of differentβ-apo-13-carotenone bacterial carotenoid oxy β-ionone (Nostoc ) +and Apo-8,10′ Microcystis PCC 7806 sp.substrate PCC 7120 specificitytuberculosis PCC 7120 Torulene 4-oxo-β-apo-8′-carotenal Torulene β-apo-8′-carotenal PpCCO Unknown β-apo-13-carotenone ) differentβ-apo-13-carotenone ( bacterial ) β-a -apocarotene-dial Torulene Table 2. Enzimatic activity substrate specificity( of caroteno Dihydroxylycopene β-ionone ( () +)and Apo-8,10′ NSC3 Transretinal ( ) 4-oxo-retinal (slow) PpCCO Unknown β-apo-8′-carotenal β-apo-14′-carotenal NSC3 Diox1 carote Other minority products Transretinal ( ) Torulene Retinal ( ) NSC3 Dihydroxylycopene Transretinal ( ) β-apo-13-carotenone ) Products ( ) -apocarotene-dial ( ) Enzymes (and Dihydroxylycopene NSC3 Transretinal PpCCO Unknown( )Substrates ( , ) Transretinal ( ) NSC3 Transretinal ( ) β-apo-14′-carotenal Other(NSC1) minority products Retinal ( ) Enzymes Assayed Substrates β-carotene-oxygenase NosCCD MtCCO Mycobacterium NSC3 Nostoc sp.PCC 680 NA Synechocystis sp. 4,4′-diapotorulene and[29] Products β-apo-10′-carotenal Torulene Torulene (References , ) 3-OH-β-ionone ( ) 4,4′-diapotorulene Astaxanthin ) Microcystis PCCUnknown 7806 Nostoc 7120 PCCTransretinal 7120 Nostoc tuberculosis Novosp Int. J. Mol. Sci. 2016, 17, 1781 9 (of 37 ( ) PpCCO Assayed Substrates β-ionone ) +PCC Apo-10,10′ β-carotene-oxygenase (NSC1) β-apo-13-carotenone MtCCO Mycobacterium NSC3 sp. NSC3 Apo-14′-diapotorulenal ( )(sp.NosCCD PpCCO Unknown 4,4′-diapotorulene β-apo-4′-carotenal 3-OH, 4-oxo-β-inone ( ) 4,4′-diapotorulene Torulene NSC3 Transretinal ( ) β-apo-10′-carotenal NSC3 Int. J. Mol. Sci. 2016, 17, 1781 : Enzyme cleaves at the C15–C15′ double bond, or equiva ) Transretinal ( β-apo-13-carotenone NSC3 Apo-14′-diapotorulenal ( ) (Nostoc β-ionone ) + Apo-8,10′ -apocarotene-dial )Apo-10,10′ Microcystis PCC 7806 7120 PCC 7120 4,4′-diapotorulene Retinal (tuberculosis ) β-apo-13-carotenone ( ) β-ionone ( sp.)( +PCC β-apo-8′-carotenal Apo-10,10′-apocarotene Retinal β-apo-13-carotenone ( ) Transretinal ( -apocarotene-dial ) ( β-ionone ( ) NSC3 Apo-14′-diapotorulenal ) β-a Int. J. Mol. Sci. 2016, 17, 1781 β-apo-13-carotenone ( () +)Apo-8,10′ Apo-12′4,4′-diapotorulene-4-al ( ) Retinal ( ) -dial ( ) NSC3 β-apo-8′-carotenal Apo-14′-diapotorulenal )-apocarotene-dial 3-OH-β-ionone ( products ) + Apo3-OH-β-apo-8′-carotenal 4,4′-diapotorulene β-apo-14′-carotenal Other( minority Retinal ( ) 4,4′-diapotorulene-4-al β-apo-13-carotenone ( ) ( carotenoid ) ( ) carote ( ) -apocarotene-dial (3-OH-β-apo-13-carotenone ) NSC3 cantaxanthinal Apo-14′-diapotorulenal ( ))activity 0 Table 2. Enzimatic and substrate specificity of different bacterial 2.2.2. CCDs with Symmetrical Mode of Action NSC3 Apo-10′-diapotorulenal ( Torulene References [19] [30,33] [36] [31] [37] [38 8,10′-apocarotene 4,4 -diapotorulene ( , minority ) 4,4′-diapotorulene Transretinal ( ) Torulene ( products ) + Apo- 3-OH-retinal NSC3 3-OH-β-apo-8′-carotenal Apo-14′-diapotorulenal 4,4′-diapotorulene-4-al Other Retinal ( )( ) Apo-10′-diapotorulenal (( 3-OH-β-ionone )) 3-OH-β-ionone ( ) NSC3 Apo-13′- caroteno Astaxanthin 3-OH-β-apo-13-carotenone ( ) β-apo-14′-carotenal β-apo-8′-carotenal -dial8,10′-apocarotene ( ) Table Enzimatic activity and substrate specificity of different bacterial 0 -diapotorulenal Int.Symbols J. Mol. Sci.indicate 2016, 17, 1781 β-apo-10′-carotenal NSC3 double bonds where oxygenases cleave. : 2.C7–C8; : C15–C15′; : C13′–C14′; : C1 Apo-14 Apo-14′-diapotorulenal ) : C9–C10; ( , ) ( ): C13–C14; Transretinal ( ) Apo-10′-diapotorulenal ( Int. J. Mol. Sci. 2016, 17, 1781 3-OH, 4-oxo-β-inone ( ) NSC3 astaxanthinone ( ) from Microcystis, β-apo-13-carotenone ( )Enzymes 3-OH-retinal ( not ) identified β-ionone ( ) + (Apo-10,10′ NSC3 TransretinalA ( β-carotene ) 4,4′-diaponeurosporene 3-OH-β-apo-10′-carotenal and Productsyet, is abl 4,4′-diapotorulene-4-al 3-OH-β-ionone )oxygenase + Apo-dial ( ) Retinal Transretinal ( ) 4,4′-diapotorulene-4-al β-apo-10′-carotenal 3-OH-β-apo-13-carotenone ) 37 means Apo-10,10′-apocarotene Apo-14′: C7′–C8′. When an enzyme cleaves atNSC3 different positions, the predominant product indicated inRetinal bold. of(of bold no product pre 4,4′-diapotorulene-4-al 4,4′-diaponeurosporene -apocarotene-dial () is )Apo-10,10′ ( Lack ) at8 β-apo-13-carotenone ( ) 10,10′-apocarotene Assayed Substrates β-ionone ( + β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. cleavages on β-carotene and zeaxanthin the C7–C8 and C7′–C8′ d NSC3 Apo-14′-diaponeurosporenal ( ) 3-OH-β-apo-10′-carotenal Enzymesastaxanthinal and Products ( ) + Apo- 3-OH-retinal ( ) -dial ( ) ( ) 4,4′-diapotorulene-4-al NSC3 Apo-10′-diapotorulenal ( 3-OH-β-ionone ) 4,4′-diaponeurosporene 3-OH-β-apo-13-carotenone ( ) NSC3 Apo-14′-diaponeurosporenal () -apocarotene-dial ) ( sp.) PCC 7120 ( ) not available. -dial Microcystis PCC 7806 PCC 7120 tuberculosis Nov Retinal ( ) NSC3 Apo-10′-diapotorulenal ( Nostoc 10,10′-apocarotene Assayed and Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc 4,40 -diapotorulene-4-al (bacterial ) +molecule Apo3-OH-β-apo-8′-carotenal suggested that one of crocetindial two molecules ofsp. β-cyc References [19] 2. Enzimatic [30,33] [36]specificity [31] [37] [38] and [38] Table activity substrate of3-OH-β-ionone different carotenoid oxygenases. NSC3 Apo-10′-diapotorulenal ( ) 3-OH-β-apo-13-carotenone ( ) 3-OH-retinal ( ) NSC3 Int. J. Mol. Sci. 2016, 17, 1781 Apo-14′-diaponeurosporenal ( ) 4,4′-diapotorulene β-apo-13-carotenone β-ionone ( -dial )sp. + Apo-8,10′ β-apo-10′-carotenal γ-carotene Int. J. Mol. Sci.Astaxanthin 2016, 17, 1781 (PCC ) 7120 Microcystis PCC Nostoc PCC 7120 tuberculosis 4,4′-diapotorulene 3-OH-β-ionone ( 7806 ) 8,10′-apocarotene 4,4′-diaponeurosporen-4′-al 0β-ionone β-apo-8′-carotenal 3-OH-β-ionone (( )[19]. + Apo-On 3-OH-β-apo-8′-carotenal 4,4′-diaponeurosporene (C15–C15′; ) + Apo-8,10′ β-a Symbols indicateand double bonds where oxygenases : C7–C8; : C9–C10; :Apo-10 C13–C14; :-dial :( C11′-C12′; C9′–C10′; of croc 3-OH-retinal )hand, Table 2. Enzimatic activity substrate specificity of different bacterial carotenoid oxygenases. molecule of the other -diapotorulenal β-apo-13-carotenone ( ) one 3-OH-β-apo-13-carotenone ( ) :molecule ( )): C13′–C14′; Int. cleave. J. Mol. Sci. 2016, 17,NSC3 1781 4,4′-diaponeurosporen-4′-al Apo-14′-diaponeurosporenal ( β-carotene ) ( ) ( ) + Apo-8,10′ β-apo-13-carotenone 3-OH, 4-oxo-β-inone ( ) -apocarotene-dial β-ionone γ-carotene Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 ( ) Retinal 8,10′-apocarotene NSC3 -apocarotene-dial ()products )Apo-8,10′ carote β-apo-8′-carotenal 4,4′-diaponeurosporene Apo-14′-diaponeurosporenal ( ) NSC3 β-apo-14′-carotenal β-ionone ( + ca Other minority NSC3 Apo-14′-diapotorulenal ( ) Retinal ( ) 4,4′-diaponeurosporen-4′-al 3-OH-retinal ( ) Apo-10′-diaponeurosporenal ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in3-OH-β-ionone bold. Lack of means preference or information hydroxyl-β-cyclocitral would beproduct released from a( molecule of β-apo-13-carotenone )bacterial ( zeaxanth ) Apo-10,10′-apocarotene 4,4′-diaponeurosporene 3-OH-β-apo-10′-carotenal ( ) no NSC3 Table 2. Enzimatic and substrate specificity of different carotenoid ox NSC3 Enzymes and ( -dial )bold +Products ApoApo-14′-diapotorulenal (activity ) -apocarotene-dial ( ) Apo-14′-diaponeurosporenal ( ) Apo-10′-diaponeurosporenal (( -apocarotene-dial )) ( , ) 4,4′-diaponeurosporene (3-OH-β-apo-13-carotenone ) NSC3 Transretinal ( ) Apo-14′-diaponeurosporenal β-apo-14′-carotenal -dial ( ) Other minority products NSC3 Retinal β-apo-13-carotenone (( of ) ) different β-carotene 10,10′-apocarotene not available. 3-OH-β-apo-10′-carotenal Assayed Substrates Table 2. Enzimatic activity and substrate specificity bacterial caroteno β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyx 0 3-OH-β-ionone ( ) + ApoEnzymes and Products Apo-12′4,4′-diaponeurosporen-4′-oic acid Apo-10′-diaponeurosporenal ( ) 2 × β-cyclocitral[30,33] ( , ) Apo-10,10′-apocarotene 3-OH-retinal ( ) 4,44,4′-diaponeurosporen-4′-al -diaponeurosporene β-apo-10′-carotenal 3-OH-β-apo-13-carotenone ( ) No ,[36] ) References [19] [37] Transretinal ( ) β-apo-12′-carotenal -dial Retinal ( [31] ) cantaxanthinal NSC3 Apo-14′-diaponeurosporenal ( 10,10′-apocarotene ) (( )))(+ Apo-10,10′ 3-OH-β-ionone acid Astaxanthin β-apo-13-carotenone ( )( and β-apo-13-carotenone ) ( Products β-carotene ) Enzymes Microcystis 7806 Nostoc NSC3 sp. PCC 7120 sp. Crocetindial PCC 7120 PpCCO tuberculosis Novosphingobium alaskensis ( , ) (( ,) )β-ionone -dial Apo-14′-diaponeurosporenal β-carotene-oxygenase NosCCD (NSC1) 4,4′-diaponeurosporen-4′-oic MtCCOPCC Mycobacterium Nostoc SaCCO Plesiocystis NSC3 Apo-10′-diaponeurosporenal ((Apo-10,10′-apocarotene ))) ( Sphingopyxis NSC3 Apo-14′-diaponeurosporenal 2 × NACOX1 β-cyclocitral 4,4′-diapotorulene-4-al 3-OH-retinal ( ) 4,4′-diaponeurosporen-4′-al 0 -diaponeurosporenal β-apo-10′-carotenal NSC3 γ-carotene 3-OH, 4-oxo-β-inone ) NosCCD 4,4′-diaponeurosporen-4′-oic acid ( double 4,4′-diapotorulene-4-al NSC3 Int. J. Mol. Sci.Astaxanthin 2016, 17, 1781 9 of 37 -dial ( Apo-10,10′ )( )( ):β-apo-14′-carotenal Symbols indicate bonds where oxygenases cleave. : (C7–C8; : (NSC1) C9–C10; C13–C14; : C13′–C14′; : -apocarotene-dial No Apo-14 Retinal () )Apo-13′) Enzymes Retinal Retinal ( : ( C15–C15′; 3-OH-β-ionone ) Assayed Substrates β-carotene-oxygenase MtCCO Mycobacterium NSC3 Nostoc sp. N NSC3 Apo-10′-diaponeurosporenal ( ) β-apo-13-carotenone ( ) β-ionone ( ) + and Products Apo-14′-diaponeurosporenal ( ) Crocetindial ( , β-ionone ) -dial ( ) ( alaskensis ) +β-apo-13-carotenone Apo-8,10′ β-a Microcystis PCC 7806 Nostoc sp. PCC 7120 7120 Apo-12′+ Apo-8,10′ tuberculosis β-ionone ( ) PCC Novosphingobium pacifica Apo-10,10′-apocarotene NSC3 β-apo-14′-carotenal ) ( means 3-OH, 4-oxo-β-inone ) astaxanthinone ) NSC3 β-apo-8′-carotenal Microcystis PCCthe 7806 Nostoc PCC (7120 PCC 7120no γ-carotene tuberculosis Novos NSC3 Apo-10′-diaponeurosporenal (( -apocarotene-dial )sp.product ( ) Retinal ( )of( bold : C7′–C8′. When an( -apocarotene-dial enzyme cleaves at different positions, predominant indicated in bold. Lack product p Assayed Substrates Apo-10′-diaponeurosporenal β-carotene-oxygenase MtCCO Mycobacterium Nostoc sp. -apocarotene-dial carote 3-OH-β-ionone ( ) (NSC1) )) Apo-8,10′ +(is Apoβ-apo-13-carotenone ((( )β-ionone 3-OH-β-apo-8′-carotenal )3-OH-β-apo-13-carotenone NSC3 Apo-10′-diapotorulenal ) )NosCCD ( ) ( ) β-apo-13carotenal ( ) ( ) ( + 2 × hydroxi-βMyxol β-apo-13-carotenone -dial ( ) Apo-12′β-ionone ( ) + Apo-8,10′ 4,4′-diaponeurosporen-4′-oic acid Apo-10,10′-apocarotene Apo-14′NSC3 Apo-10′-diapotorulenal ) 3-OH-β-apo-13-carotenone 4,4′-diaponeurosporen-4′-al β-apo-13-carotenone Zeaxanthin β-ionone ( ) + Apo-8,10′ 3-OH-β-ionone ( PCC ) Microcystis PCC 7806 Nostoc sp. 7120 7120 tuberculosis Myxol 8,10′-apocarotene not available. 3-OH-β-apo-14′-carotenal (Apo-12′) ( ) ( ( ) ) [37]PCC 4,4′-diaponeurosporen-4′-al 3-OH-β-ionone ( ) 3-OH-β-ionone -apocarotene-dial ( Apo) cyclocitral β-apo-14′-carotenal References [19] [30,33] [36] [31]astaxanthinal ( ) + 3-OH-β-apo-8′-carotenal carotenone Apo-10′minority products β-apo-13-carotenone ( )β-apo-8′-carotenal ( NSC3 ) 3-OH-β-apo-13-carotenone Retinal ( ) (3R)-3-OH-β-apo-8′-carotenal -dial acid ( 0)-al Other 2 × hydroxi-ββ-apo-13carotenal ( ) -apocarotene-dial 4,4 ( 4,4′-diaponeurosporen-4′-oic )0 -diaponeurosporen-4 β-apo-13-carotenone ( ) ( ) β-apo-13-carotenone ( ) 3-OH-retinal ) β-carotene β-apocarotene-dial ( ) NosCCD No Apo-10,10′-apocarotene Apo-10′-diaponeurosporenal ( ) -dial (( )) + Apo-8,10′ cantaxanthinal 3-OH-β-apo-13-carotenone ( () )β-apo-13-carotenone Zeaxanthin ( ) ( β-ionone Myxol ( ) 2 × β-cyclocitral ( , )0 Apo-10,10′-apocarotene Apo-8′-10-apocarotenedial ( , 3-OH-β-ionone )[31] 3-OH-retinal ( ) Apo-14′-diaponeurosporenal ( )3-OH-β-ionone )8,10′-apocarotene β-apo-8′-carotenal (β-apo-14′-carotenal ) No NosCCD cyclocitral References Other minority products [19] [30,33] [36] [37] ( β-carotene , ) where carotenal ( caro ): Other minority products (( )):( [38] )3-OH-β-apo-14′-carotenal β-apo-14′-carotenal Retinal ( : Transretinal )C9–C10; Retinal ( ( ) ): C15–C15′; carotenone ( -diaponeurosporenal ): C7–C8; Symbols indicate bonds cleave. C13–C14; : C13′–C14′; Apo-14 NSC3 oxygenases Retinal ( ) ( double 3-OH-β-ionone ) 3-OH-β-apo-10′-carotenal Apo-13′Astaxanthin β-apo-13-carotenone ) (No) β-apo-13-carotenone NSC3 Apo-10′-diaponeurosporenal 3-OH-retinal ( [38] )( 3-OH-retinal -apocarotene-dial Apo-14′-diaponeurosporenal ((Apo-10,10′-apocarotene 3-OH-β-ionone ) ( -dial Apo-8′-10-apocarotenedial ) )) Apo-10′( ( , , ) ) ( , ( 3-OH-β-ionone ( -dial ) ( ( ) +) Apo3-OH-β-apo-11-carotenal Apo-10′-diaponeurosporenal (Apo-10,10′-apocarotene NSC3 Crocetindial 4,4′-diaponeurosporene NSC3 NosCCD 2 × β-cyclocitral ) -dial 3-OH-retinal (? )( ) Transretinal ()): C13′–C14′; ,minority ) 0 3-OH-β-apo-13-carotenone β-apo-14′-carotenal ( ) 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) β-apo-14′-carotenal Other products ( bonds , ) Apo-8′-10-apocarotenedial ( ) β-apo-10′-carotenal Apo-10′-diaponeurosporenal ( carotenal ( ) No)product Symbols indicate double where4,4′-diaponeurosporene oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C11′-C12′; : C9′–C10′; Retinal ( ) of bold means (no -dial ( ) is (indicated Transretinal ( ) Apo-10 -diaponeurosporenal ) : C7′–C8′. When an enzyme cleaves at different positions, the( predominant product in bold. Lack Myxol Myxol-2′-fucoside 10,10′-apocarotene 3-OH-β-apo-10′-carotenal -dial ( ) Crocetindial , ) 3-OH-β-ionone ( ) + Apo3-OH-β-apo-11-carotenal ? β-apo-13-carotenone ( ) β-ionone ( ) + Apo-10,10′ β-apo-10′-carotenal 3-OH-retinal ( Apo-14′) Apo-10,10′-apocarotene ) 3-OH-β-apo-13-carotenone Myxol-2′-fucoside β-apo-14′-carotenal 3-OH-β-ionone (bold ) -dial(no ( ,)product Lycopene Myxol β-apo-13-carotenone ( ) ( ) ( ) Transretinal ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack ofβ-ionone preference or information not available. 3-OH-β-ionone ) means NSC3 NosCCD Apo-14′-diaponeurosporenal ( (10,10′-apocarotene ) ) + Apo-10,10′ 3-OH-β-apo-13-carotenone (( ) ) 2Apo-14′-diaponeurosporenal ×Apo-8′-10-apocarotenedial hydroxi-β-dial acid ( ) astaxanthinal ( ) No ( ) Myxol-2′-fucoside β-apo-13-carotenone ( -apocarotene-dial ) Zeaxanthin Retinal ( ) (( 3-OH-β-ionone NSC3 β-apo-10′-carotenal β-ionone ( ) + Apo-10,10′ 3-OH-retinal 4,4′-diaponeurosporen-4′-oic 3-OH-β-ionone ) (( ( ))) ( (() )) (3R)-3-OH-β-apo-12′-carotenal 0 -diaponeurosporen-4 0 -oic -apocarotene-dial γ-carotene Retinal ( ) -dial Lycopene NosCCD Apo-8′-10-apocarotenedial β-apo-13-carotenone ( ) β-ionone ( ) + Apo-10,10′ 4,4′-diaponeurosporen-4′-oic acid not available. 3-OH-β-apo-14′-carotenal ( ) 4,4 acid NosCCD cyclocitral References [30,33] [36] [37]( ) + Apo-8,10′ [38]3-OH-β-apo-13-carotenone [38] ( ) 2 ×[31] hydroxi-β- Apo-10,10′-apocarotene No Apo-8′-10-apocarotenedial )3-OH-β-ionone No -apocarotene-dial ( [19] ) Retinal ( ) 3-OH-β-ionone ( β-ionone ) ( -apocarotene-dial NosCCD Zeaxanthin ( (3-OH-β-apo-13-carotenone )) acetate adduct C10 () ) γ-carotene Retinal ( ( , ) 3-OH-retinal ( ) 3-OH-retinal 3-OH-β-ionone ( ) + Apo3-OH-β-apo-8′-carotenal 3-OH-β-apo-14′-carotenal ( ) 0 3-OH-β-ionone ( )( ): C11′-C12′; ca cyclocitral Myxol-2′-fucoside -dial )(( ))++ApoSymbols indicate double bonds where oxygenases cleave. : 3-OH-β-apo-8′-carotenal C7–C8; : adduct C15–C15′; : C9′–C10′; NSC3 : C13–C14; Apo-8,10′ NosCCD Apo-8′-10-apocarotenedial ( β-ionone ) )): (C13′–C14′; acetate C10 -apocarotene-dial Apo-10 NSC3 Apo-10′-diaponeurosporenal 3-OH-β-apo-13-carotenone 3-OH-β-apo-13-carotenone ((Apo-10,10′-apocarotene No 3-OH-β-apo-14′-carotenal ( ? () ) ( ) Lutein : C9–C10; Crocetindial ( ( , , ) ) -diaponeurosporenal 3-OH-β-apo-11-carotenal 3-OH-β-apo-13-carotenone 4,4′-diaponeurosporen-4′-al 8,10′-apocarotene NSC3 Apo-10′-diaponeurosporenal ( ) 3-OH-retinal ( ) 8,10′-apocarotene Myxol-2′-fucoside 3-OH-β-ionone (cleaves ) + Apo( ) ) +( Apoal -apocarotene-dial ) preference acetate adduct C10) 3-OH-β-ionone 3-OH-β-apo-8′-carotenal 3-OH-β-ionone -dial (product 3-OH-retinal Int. J. Mol. Sci. 2016, 17, 1781 11 of 38 : C7′–C8′. When an enzyme at4,4′-diaponeurosporen-4′-al different positions, the predominant product in bold. Lack of no or( (information 3-OH-retinal ) ) ( ) ( ( )) β-apo-13-carotenone 3-OH-β-apo-13-carotenone 3-OH-retinal ( (bold ) means 3-OH-β-apo-13-carotenone ( ) β-carotene 3-OH-β-apo-14′-carotenal Lutein is indicated 4-ketomyxol-2′-fucoside -dial ( ) Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? 2 ×Apo-8′-10-apocarotenedial β-cyclocitral ( , )( )Apo-10,10′-apocarotene -dial ( NosCCD ) 8,10′-apocarotene ( ( ) )8,10′-apocarotene 3-OH-α-apo-15′-carotenal 3-OH-β-ionone Apo-14′-diaponeurosporenal 4-ketomyxol-2′-fucoside No Lycopene Retinal ( ) ( )( ) 3-OH-retinal β-apo-13-carotenone not available. β-carotene Apo-14′-diaponeurosporenal ( ) -dial NSC3 3-OH-retinal ( )3-OH-β-apo-10′-carotenal ( ( ) )+( ApoCrocetindial , ( Apo-8′-lycopenal ) , 3-OH-β-ionone -dial ) No acetate( adduct C10 NSC3 2 × β-cyclocitral Apo-8′-10-apocarotenedial )) 3-OH-β-apo-13-carotenone 3-OH-β-apo-10′-carotenal Myxol Apo-10′-diaponeurosporenal -dial ( ) 4-ketomyxol-2′-fucoside 3-OH-β-ionone ( ) ) ( ((Apo-10,10′-apocarotene ( )Retinal ( ()( )) 3-OH-β-ionone ( NosCCD ) + Apoβ-apo-14′-carotenal 3-OH-α-apo-15′-carotenal Lycopene Apo-10′-diaponeurosporenal )) 10,10′-apocarotene Myxol NosCCD 3-OH-β-apo-13-carotenone ( 3-OH-4-oxo-β-ionone ( ) 3-OH-β-apo-10′-carotenal 3-OH-β-ionone ( ) 3-OH-β-ionone ( ) + ApoCrocetindial (3-OH-β-ionone , C10 )4-oxo-β-ionone -dial ( ) AcycloretinalNo (slow) acetate adduct No 3-OH-retinal Apo-8′-10-apocarotenedial ) NosCCD Myxol NosCCD 3-OH-β-apo-13-carotenone al 10,10′-apocarotene (( )) ( ) ( ) ( ) 3-OH-4-oxo-β-ionone ( () -dial 3-OH-β-apo-13-carotenone β-apo-14′-carotenal 3-OH-β-ionone ( ) + Apo( )(( )) Apo-8′-10-apocarotenedial Apo-8′-10-apocarotenedial 10,10′-apocarotene NosCCD NosCCD 3-OH-retinal ( Apo-10,10′-apocarotene )( ((() )))4-oxo-β-ionone 3-OH-β-apo-13-carotenone ( Echinenone ) 0 -10-apocarotenedial 3-OH-β-apo-13-carotenone 23-OH-4-oxo-β-ionone × hydroxi-β4-ketomyxol-2′-fucoside 3-OH-retinal ( Apo-8′-10-apocarotenedial 3-OH-β-apo-14′-carotenal ( ) )( ) ( ) ( ) Apo-8 ( ) Lutein -dial ( ) Zeaxanthin No γ-carotene 10,10′-apocarotene 4,4′-diaponeurosporen-4′-oic acid 3-OH-β-ionone 3-OH-β-apo-13-carotenone -dial ( () ) Echinenone NosCCD 3-OH-β-apo-14′-carotenal ( ) -dial ( ) 4,4′-diaponeurosporen-4′-oic acid cyclocitral β3-OH-retinal ( ) 4-ketomyxol-2′-fucoside Apo-8′-10-apocarotenedial ( Apo-10,10′-apocarotene )( ) + Apo-8,10′ 3-OH-β-apo-13-carotenone 2 × hydroxi-β- β-ionone 3-OH-retinal ( ) No Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal (( )) Lutein Zeaxanthin γ-carotene γ-carotene-dial ( ) 3-OH-β-ionone ( ) Apo-10′-lycopenal β-ionone (-dial ) ( cyclocitral , No ) 3-OH-retinal ( ) -apocarotene-dial caro 3-OH-β-apo-14′-carotenal ( ) ) 3-OH-α-apo-15′-carotenal 3-OH-4-oxo-β-ionone ( β-ionone ) (( ))((+ Apo-8,10′ -dial β-ionone ( ) + Apo-8,10′ β-apo-133-OH-retinal ( ) No 0 -fucoside 4-hydroxymyxol-2′-fucoside NSC3 Apo-10′-diaponeurosporenal (Apo-10,10′-apocarotene ) β-ionone ( ) Canthaxantin NosCCD Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? Myxol-2′-fucoside Myxol-2 NSC3 Apo-10′-diaponeurosporenal ( , ) ( )No information Apo-8′-10-apocarotenedial ()( -apocarotene-dial )) ( ) ( 3-OH-retinal ) 3-OH-4-oxo-β-ionone 3-OH-α-apo-15′-carotenal -apocarotene-dial ( ) carotenone -dial(( )) β-apo-13-carotenone ( ) ( ) β-carotene NosCCD β-ionone ( ) + Apo-8,10′4-hydroxymyxol-2′-fucoside β-apo-13-( , 2)x (4-oxo-β-ionone Canthaxantin Crocetindial 3-OH-β-apo-11-carotenal ? 2 × β-cyclocitral ( , 3-OH-β-ionone ) Apo-10,10′-apocarotene 4-oxo-β-ionone Apo-8′-10-apocarotenedial ( ) (( )) 4-hydroxymyxol-2′-fucoside No Echinenone Retinal ( () ) Lycopene β-apo-13-carotenone ( ) β-carotene -apocarotene-dial ( ) carotenone ( )0 ) Apo-10,10′-apocarotene 3,3-OH-β-ionone 4-OH-β-ionone ( ) ) 2-dial Crocetindial ( , ( ) x 4-oxo-β-ionone ( ) Apo-10,10′-apocarotene ( 2 ×No β-cyclocitral , ( )( ) ) Apo-10,10′-apocarotene No NosCCD 4-oxo-β-ionone (( )) β-apo-14′-carotenal ( ) NosCCD β-apo-13-carotenone Apo-8 (-10-apocarotenedial β-carotene 3,3-OH-β-ionone 4-OH-β-ionone No Echinenone Retinal ( ) Apo-8′-10-apocarotenedial )) (( )) ( ) (( -dial Lycopene β-apo-8′-carotenol Apo-10,10′-apocarotene NosCCD -dial NosCCD Apo-8′-10-apocarotenedial 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene 3-OH-β-ionone ( ) Crocetindial ( , ) -dial ( ) Myxol NosCCD 4-hydroxymyxol-2′-fucoside 3,Retinal 4-OH-β-ionone ( )adduct Apo-8′-10-apocarotenedial (( Apo-10,10′-apocarotene )) C10 No (( )) NoNo (in vitro) Myxol NosCCD (acetate ) C10 β-apo-13-carotenone ( ) β-apo-14′-carotenal Apo-8′-10-apocarotenedial Noadduct 3-OH-β-apo-13-carotenone NosCCD β-ionone (-dial ) (( ))No acetate Apo-8′-10-apocarotenedial ( ) Crocetindial ( , ) -dial ( ) -dial Retinal 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene4-hydroxymyxol-2′-fucoside 3-OH-β-apo-13-carotenone ( ) 2Apo-8′-10-apocarotenedial × hydroxi-β( ) 3-OH-β-apo-14′-carotenal ( ) Zeaxanthin β-apo-14′-carotenal ( 3-OH-β-ionone ) No β-ionone Lutein No ( )( ) No (in vitro) No Canthaxantin Retinal ( ) 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-14′-carotenal ( ) cyclocitral Crocetindial ( , ) -dial ( ) 3,24-OH-β-ionone ( ) 3-OH-β-apo-13-carotenone ( ) × hydroxi-β- Apo-10,10′-apocarotene No 3-OH-retinal ( ) 4-oxo-β-ionone ( ) ( ) 3-OH-β-apo-14′-carotenal ( ) NosCCD Zeaxanthin Lutein 3-OH-β-ionone β-apo-14′-carotenal ( ) Canthaxantin 0 -fucoside ) Symbols indicate double bonds where oxygenases cleave. : C9=C10; Apo-8′-10-apocarotenedial :( C13=C14; :2 (xC15=C15′; 3-OH-retinal ( ) ) 3-OH-β-apo-14′-carotenal ( ) cyclocitral 3,, 4-OH-β-ionone ) ( -dial 4-ketomyxol-2 ( ) 3-OH-α-apo-15′-carotenal Apo-10,10′-apocarotene Symbols indicate4-ketomyxol-2′-fucoside double bonds where oxygenases cleave. : C9=C10; : C13=C14; : C15=C15′; No 3-OH-retinal ( ? ) NosCCD 3-OH-β-apo-13-carotenone (Apo-10,10′-apocarotene 2 × hydroxi-β2 )x) 4-oxo-β-ionone ( )3-OH-β-apo-11-carotenal Crocetindial ( ,, )) (3R)-3-OH-β-apo-8′-carotenol Apo-13′( Apo-8′-10-apocarotenedial ( 3-OH-retinal ( ) : C13′=C14′; : C11′=C12′; : C9′=C10′; :3-OH-β-ionone C7′=C8′.cleave. ( ): C9=C10; : C13=C14; Zeaxanthin ( ) ) Symbols indicate double bonds where oxygenases : C15=C15′; ( ) 3-OH-α-apo-15′-carotenal Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal (-dial ) ( -dial : C13′=C14′; : C11′=C12′; : C9′=C10′; : (C7′=C8′. cyclocitral 3-OH-β-apo-13-carotenone ) Crocetindial ( , ) zeaxanthinone ( ) 2 × hydroxi-β3-OH-β-apo-11-carotenal ? 3-OH-retinal 4-oxo-β-ionone ( )No )) 3-OH-4-oxo-β-ionone ( No No (in vitro) Apo-10,10′-apocarotene ( ) 3-OH-β-ionone ( ) -dial ( EchinenoneNosCCD : C13′=C14′; : C11′=C12′; ( , ): C9′=C10′; (: C7′=C8′. 3-OH-retinal ( ) Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal ) Lycopene Apo-14′cyclocitral 0 -10-apocarotenedial No Symbols indicate double where oxygenases cleave. : C13=C14; : C15=C15′; 4-oxo-β-ionone Int. J. bonds Mol. Sci. 2016, 17, 1781-dial ( )No : C9=C10; 3-OH-4-oxo-β-ionone Apo-8 No (in vitro) (( )) Apo-10,10′-apocarotene No Int. Int. J. Mol. Sci. Sci. 2016, 17, 1781 J. Mol. 2016, 17, 1781 ( )? No Lycopene Echinenone Crocetindial ( , ) -dial ( ) 3-OH-β-apo-11-carotenal zeaxanthinal ( ) ( , ) ( oxygenases ) NosCCD No Symbols double3-OH-retinal bonds where cleave. : C9=C10; Apo-8′-10-apocarotenedial : C13=C14; : C15=C15′; ( Apo-10,10′-apocarotene )β-ionone ( ) : C13′=C14′; : C11′=C12′; C9′=C10′; : C7′=C8′. -dial ( ) indicate 3-OH-β-apo-13-carotenone ( ) No 9 of :37 Apo-8′-lycopenol -dial ( ) Apo-12′Crocetindial ( , ) 3-OH-β-apo-11-carotenal 3-OH-β-apo-14′-carotenal ( ) ( ) : C13′=C14′; : C11′=C12′; : C9′=C10′; : ?C7′=C8′. Lutein 3-OH-β-apo-13-carotenone Canthaxantin β-ionone ( ) No information Lycopene zeaxanthinal Apo-12′Apo3-OH-retinal ( () ) 3-OH-β-apo-14′-carotenal ( ) Lutein 2 x 4-oxo-β-ionone No ( ) 0 -fucoside No poorly Canthaxantin 4-hydroxymyxol-2 cantaxanthina 1 cantaxanth 3-OH-α-apo-15′-carotenal Apo-12′Yes3-OH-retinal (in vivo) ( ) Apo-10,10′-apocarotene 2 x 4-oxo-β-ionone ( ) No No poorly 3-OH-β-ionone ( ) Astaxanthin ( ) product 3-OH-β-ionone ( )( ) Apo-13′cantaxanthinal ( 3-OH-β-ionone ) 4-hydroxymyxol-2′-fucoside ApoAstaxanthin 3-OH-α-apo-15′-carotenal Astaxanthin Unknown 3-OH-β-apo-13-carotenone (Apo-10,10′-apocarotene ) -dial ( ) 4-oxo-β-apo-8′-carotenal 3-OH,4-oxo-β-ionone 4-oxo-β-inone 3, 4-OH-β-ionone (( ))( ) ( ) 3-OH, 4-oxo-β-inone ( () (Echinenone astaxanthinone 3-OH, 4-oxo-β-inone astaxanthi Apo-13′( ) -dial ( ) Lutein 3-OH-β-apo-13-carotenone ) ) NosCCD 3-OH-β-apo-14′-carotenal 4-oxo-retinal (slow) 0 Apo-10,10′-apocarotene Apo-10,10′-apocarotene 4-oxo-β-ionone (( )) Apo-10,10′-apocarotene Apo-14′NoApo-8 -10-apocarotenedial Apo-10,10′-apocarotene Apoastaxanthinone ( ) Echinenone 3-OH-retinal ( ) Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal ( ) No -dial ( ) 3, 4-OH-β-ionone ( ) -dial ( ) -dial-dial ( )( ) astaxanthinal astaxanth Apo-14′-3-OH-retinal No NosCCD β-ionone-dial ( )( ) 3-OH-α-apo-15′-carotenal ( ) No Apo-8′-10-apocarotenedial ( [30,33] ) References [36] [38] [38] [31] References [29] References [19] [19]astaxanthinal [36] [36] [19] [38] [38 ( ) [30,33] References [30,33] Canthaxantin β-ionone ( )[37] [37] ( ) [31] [31] 3-OH-α-apo-15′-carotenal 2 xcleave. 4-oxo-β-ionone0:( C7–C8; ) 1] [37] [38] [38] Canthaxantin 0 0 Symbols indicate double bonds where oxygenases : C9–C10; : C13–C14; : C1 4-oxo-β-ionone ( : ):C9–C10; Symbols indicate double bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; C13′–C14′; : C11′-C12′; : C9′–C10 Symbols indicate double bonds where oxygenases cleave. : C7–C8; C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C : Enzyme cleaves at the C15–C15′ double bond, or equiv Symbols indicate double bonds where oxygenases cleave. C9=C10; : C13=C14; C15=C15 ; : C13 =C14 ; ( ) Apo-10,10′-apocarotene Echinenone 2 x 4-oxo-β-ionone ( ) 0 =C120 ;( ): :C9 0 =C100 ; : C7′–C8′. 0 =C80 .When Apo-10,10′-apocarotene : C15–C15′; : C13′–C14′; C11′-C12′; C9′–C10′; 4-oxo-β-ionone an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack : C11 C7 -dial ( ) : C7′–C8′. When an enzyme cleaves at different positions, the the predominant product is indicated in bold. Lack of bold means no product preference or information : C7′–C8′. When an enzyme cleaves at different positions, predominant product is indicated in bold. Lack of bold means no product preference or informa Apo-10,10′-apocarotene

Table 3. Substrates tested and cleaved with a unique oxygenase.

and a dialdehyde product (between C to C ) (Table 1). Howev β-carotene (C ), are not cleaved [29]. NosACO, also named NSC2 [30], is one of the three CCDs of (strain PCC 7120). The three enzymes exhibit different substrate cooperative functions among them [31]. In vitro, NosACO cleaves m at C15–C15′ double bonds to generate retinal. In vivo, bleaching observed on β-carotene, zeaxanthin, torulene, lycopene, and diapoca

34

33

30

34

33

34

30

13 13

33

13

39 39

30

39

No

-dial ( ) Symbols indicate double bonds where oxygenases cleave. : C9=C10; : C13=C14; : C15=C15′; -dial ( ) Apo-10,10′-apocarotene Lacknot of not bold means No no product preference or informationnot available. 2.2.2. CCDs with Symmetrical Mode of Action available. available. β-ionone ( ) -dial ( ) : C13′=C14′; : C11′=C12′; : C9′=C10′; : C7′=C8′. Canthaxantin β-ionone ( )

A β-carotene oxygenase from Microcystis, not identified yet, is ab

2 x 4-oxo-β-ionone ( ) 3. Fungal Carotenoid Oxygenases Apo-13′-cantaApo-10,10′-apocarotene cleavages on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ 2 x 4-oxo-β-ionone ( ) xanthinone ( ) Carotenoid production is a frequent trait in fungi. examples are the production of -dial ( Best-known ) Apo-10,10′-apocarotene suggested that one molecule of crocetindial and two molecules of β-cy Apo-14′β-carotene -dial ( )in different taxonomic groups, neurosporaxanthin in ascomycetes, and astaxanthin in ( one ) molecule of β-carotene [19]. On the cantaxanthinal other hand, molecule of cro

basidiomycetes. In some fungi, as Phycomyces blakesleeanus, Mucor circinelloides, Neurospora crassa, hydroxyl-β-cyclocitral would Fusarium fujikuroi, and Xanthophyllomyces dendrorhous, the genetic and biochemical basis of their be

released from a molecule of zeaxant

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3. Fungal FungalCarotenoid CarotenoidOxygenases Oxygenases 3. Carotenoid production production is is aafrequent frequenttrait traitininfungi. fungi.Best-known Best-known examples production Carotenoid examples areare thethe production of of β-carotene in different taxonomic groups, neurosporaxanthin in ascomycetes, and astaxanthin β-carotene in different taxonomic groups, neurosporaxanthin in ascomycetes, and astaxanthin in in basidiomycetes. In some fungi, as Phycomyces blakesleeanus, Mucor circinelloides, Neurospora crassa, basidiomycetes. In some fungi, as Phycomyces blakesleeanus, Mucor circinelloides, Neurospora crassa, Fusarium and Xanthophyllomyces dendrorhous, the the genetic genetic and and biochemical biochemical basis basis of of their Fusarium fujikuroi, fujikuroi, and Xanthophyllomyces dendrorhous, their carotenoid production has received considerable attention, and the functions and regulation of all the the carotenoid production has received considerable attention, and the functions and regulation of all structural genes been thoroughly investigated [2]. In[2]. addition, some fungi are fungi excellent structural geneshave have been thoroughly investigated In addition, some arecarotenoid excellent producers, and they have been adopted as biotechnological carotenoid sources [ 39,40]. In contrast to carotenoid producers, and they have been adopted as biotechnological carotenoid sources [39,40]. In photosynthetic organisms, in the cases investigated, the presence of carotenoids is dispensable, and the contrast to photosynthetic organisms, in the cases investigated, the presence of carotenoids is mutants unable make carotenoids In some fungal models,Insuch as fungal N. crassa, such mutants dispensable, andtothe mutants unableare to viable. make carotenoids are viable. some models, such as have been widely used as easily traceable genetic markers. N. crassa, such mutants have been widely used as easily traceable genetic markers. In fungi, fungi, carotenoid carotenoid biosynthesis biosynthesis derives derives from from the the mevalonate mevalonatepathway, pathway, as as indicates indicatesthe thelabeling labeling In of carotenoids with radioactive mevalonate [41,42]. The biosynthetic pathways are similar to those of of carotenoids with radioactive mevalonate [41,42]. The biosynthetic pathways are similar to those of photosynthetic species, except that a single desaturase is responsible for all desaturation steps from photosynthetic species, except that a single desaturase is responsible for all desaturation steps from phytoene, and the the cyclase cyclase and and phytoene phytoene synthase synthase activities activities depend depend on on aa single single bifunctional bifunctional gene. gene. phytoene, and Several CCD enzymes have been identified in fungi participating in late steps of their carotenoid Several CCD enzymes have been identified in fungi participating in late steps of their pathways. are related to are the production different of compounds: retinal,compounds: neurosporaxanthin, carotenoidThey pathways. They related to of thethree production three different retinal, and trisporic acids (Figure 2). neurosporaxanthin, and trisporic acids (Figure 2).

Figure 2. Enzymatic reactions achieved by fungal CCDs. (A) Retinal production from β-carotene by Figure 2. Enzymatic reactions achieved by fungal CCDs. (A) Retinal production from β-carotene by the the Cco1 (Ustilago maydis) and CarX (F. fujikuroi) CCDs; (B) β-apo-4′-carotenal production from Cco1 (Ustilago maydis) and CarX (F. fujikuroi) CCDs; (B) β-apo-40 -carotenal production from torulene by torulene by the CarT (F. fujikuroi) and Cao-2 (N. crassa) CCDs; (C) β-apo-13-carotenone production the CarT (F. fujikuroi) and Cao-2 (N. crassa) CCDs; (C) β-apo-13-carotenone production from β-carotene from β-carotene by the sequential cleavage by the CarS and AcaA CCDs (P. blakesleeanus). Cleavage by the sequential cleavage by the CarS and AcaA CCDs (P. blakesleeanus). Cleavage sites are shaded sites are shaded and indicated by an arrow. and indicated by an arrow.

3.1. Production 3.1. Fungal Fungal CCDs CCDs Involved Involved in in Retinal Retinal Production Rhodopsins are membrane membrane photoreceptors photoreceptors using using retinal retinal as as aa chromophore. Rhodopsins are chromophore. The The tertiary tertiary structure structure of these proteins is highly conserved and consists of seven transmembrane helices a lysine lysine of these proteins is highly conserved and consists of seven transmembrane helices with with a residue to which an all-trans-retinal chromophore is covalently bound. Rhodopsins are found all residue to which an all-trans-retinal chromophore is covalently bound. Rhodopsins are found in in all major taxonomic groups, few fungal fungal rhodopsins rhodopsins have have been been functionally functionally major taxonomic groups, including including fungi fungi [43]. [43]. Very Very few investigated, among them NOP-1 in N. crassa [44], Ops in Leptosphaeria maculans [45,46], and investigated, among them NOP-1 in N. crassa [44], Ops in Leptosphaeria maculans [45,46], and CarO [47], CarO [47], and OpsA in F. fujikuroi [48]. The participation of retinal in the activity of these proteins and OpsA in F. fujikuroi [48]. The participation of retinal in the activity of these proteins has only been has only been demonstrated NOP-1 demonstrated for NOP-1 [49]for and CarO [49] [50].and CarO [50]. Animals obtain retinal from β-carotene through its oxidative cleavage by a class of CCD enzymes [51]. A similar CCD, encoded by the gene carX [52], is used by F. fujikuroi to produce retinal from β-carotene (Figure 2) [53]. The gene carX is located in a coregulated cluster with the genes

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Animals obtain retinal from β-carotene through its oxidative cleavage by a class of CCD enzymes [51]. A similar CCD, encoded by the gene carX [52], is used by F. fujikuroi to produce retinal from β-carotene (Figure 2) [53]. The gene carX is located in a coregulated cluster with the genes needed to produce β-carotene, carRA, and carB, and with the rhodopsin gene carO. Unexpectedly, despite the biochemical similarities between their carotenoid pathways, no carX ortholog has been identified in N. crassa. The only possible candidate for such activity in the genome of this fungus, the CCD CAO-1, was not active on carotenoid substrates, but cleaved efficiently the stilbene resveratrol [54]. A retinal-forming CCD enzyme, called Cco1, has also been described in the basidiomycete Ustilago maydis, producing minor amounts of β-carotene [55]. In this organism, retinal could be detected in cell extracts of the wild type and strains overexpressing cco1, but not in those of null cco1 mutants, and the in vivo β-carotene levels correlated inversely with the Cco1 activity. Retinal production seems to be particularly relevant in this fungus, as suggested by the occurrence of three genes in its genome for presumptive photoactive rhodopsins [55]. 3.2. Fungal CCDs Involved in Trisporic Acid Production Trisporic acids, a class of fungal sexual hormones belonging to the trisporoids family, stand out among the compounds derived from β-carotene for their biological relevance [56,57]. Depending on small variations in their chemical structures, these hormones are distributed in five groups, named A, B, C, D, and E [56,58,59]. The synthesis, investigated in detail in B. trispora and other related species, requires the participation of two CCD enzymes. In fact, thorough chemical analyses revealed the generation of an unexpected apocarotenoid complexity in these fungi: B. trispora produces at least three groups of β-carotene derivatives—C18 trisporoids, C15 cyclofarnesoids, and C7 methylhexanoids [60]—and the same compounds were found in P. blakesleeanus cultures [61]. In the latter case, the origin from β-carotene was confirmed by their absence in a mutant unable to produce β-carotene. The first CCD enzymes known in fungi were Tsp3 and Tsp4 from Rhizopus oryzae, identified through the analysis of its genome and involved in trisporic acid biosynthesis [62]. This discovery led to identification of the tsp3 ortholog in B. trispora, whose connection with trisporic acids was reinforced by its induced expression by sexual interaction and its capacity to cleave β-carotene in a tsp3-expressing E. coli strain. Further evidence was obtained from the chemical structures of the apocarotenoids produced by P. blakesleeanus [63], which were consistent with the cleavage of β-carotene at the C110 –C120 and C12–C13 double bonds (Figure 2). The carS mutants of P. blakesleeanus, which accumulate large amounts of β-carotene, were found to be affected in a CCD enzyme [64], orthologous of Tsp3. The function of the gene, corroborated by the finding of relevant mutations in six different carS mutants, was confirmed by the capacity of the CarS protein to generate β-apo-120 -carotenal (Figure 2) in carS-expressing E. coli cells [65]. The identification of CarS as a CCD was unexpected, since the β-carotene over-accumulation phenotype of carS mutants suggested that the gene encoded a regulatory protein. This finding leads to the reinterpretation of the carotene overproduction of the carS mutation as a result of a blockage of the pathway or of the lack of a negative-acting apocarotenoid signal, which could be responsible for a formerly proposed feed-back regulation [66]. In support of the blockage hypothesis, the large increase in β-carotene content in these mutants is not sufficiently explained by the minor changes that could be found in the transcript levels of the structural genes carB and carRA [67]. However, the function of CarS as a CCD enzyme does not discard an additional regulatory role, as suggested by the unexpected albino phenotype of some double carS mutants [68]. Moreover, the carS mutants exhibit increased enzymatic activities in vitro [42], a result more coherent with a putative regulatory hypothesis. A second CCD enzyme, AcaA, has been characterized in P. blakeseleeanus. AcaA cleaves β-apo-120 -carotenal (C25 ) to generate β-apo-13-carotenone (C18 ) [65]. The available information indicates that CarS and AcaA act sequentially to produce apocarotenoids in this fungus. First, CarS cleaves β-carotene at the C110 –C120 double bond to generate the C25 and C15 apocarotenals, and AcaA

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cleaves the resulting C25 -product at the C13–C14 double bond afterwards (Figure 2). The resulting C18 product is the origin of the trisporic acids and other trisporoids, while the former CarS C15 product is used for the synthesis of cyclofarnesoids. The genome of P. blakesleeanus contains three additional genes for presumptive CCDs. Only one of them contains the expected histidine residues to support CCD cleavage activity, but its function has not been investigated. The genes and enzymes for later steps of trisporoid metabolism are currently under study, but they are presumably CCD-unrelated enzymes: they include at least a 4-dihydromethyltrisporate dehydrogenase [69,70] and a 4-dihydrotrisporin-dehydrogenase [71], both identified in the P. blasleeanus relative Mucor mucedo. 3.3. Fungal CCDs Involved in Neurosporaxanthin Production Neurosporaxanthin is a C 35 carotenoid derived from the oxidative cleavage of its C 40 precursor torulene. The first enzymatic reactions in the neurosporaxanthin biosynthetic pathway are similar to those for β-carotene production in other fungi, but in this case five desaturations instead of four and only one cyclization are introduced into the C 40 carotene skeleton, leading to torulene (Figure 2). The analysis of the proteomes of N. crassa and F. fujikuroi and the study of mutants blocked in different steps of the pathway led to the identification of the enzymes involved in the late oxidative reactions. The genomes of both species contain two CCD-encoding genes. One of them, called carT in F. fujikuroi, was found to catalyze the cleavage of torulene to produce β-apo-120 -carotenal. This function was corroborated by the finding of a mutated carT allele in a reddish torulene -accumulating mutant and the ability of the wild-type carT allele to restore neurosporaxanthin production when it was introduced in the mutant [53,72]. CarT activity was also confirmed by targeted mutation in Gibberella zeae, a teleomorph of Fusarium graminearum [73]. The same function was achieved in N. crassa by its ortholog, cao-2, as demonstrated the accumulation of torulene in a mutant for this gene and the finding of cao-2 mutations in two torulene-producing mutants of this fungus [74]. As found for β-carotene production in Mucorales, the synthesis of neurosporaxanthin is induced by the light in N. crassa [75] and Fusarium sp. [76]. This photoresponse is achieved through an outstanding increase in mRNA levels for most of the structural genes, including cao-2 [74] in N. crassa, and carT [72] in F. fujikuroi. Moreover, the expression of carT is enhanced in carotenoid-overproducing mutants. The CarT and CAO-2 enzymes, catalyzing the asymmetrical cleavage of torulene at its acyclic end to remove a C5 segment, represent a novel CCD subgroup. The reaction is highly specific, as shown by the incapacity of CAO-2 to cleave the torulene precursor γ-carotene, indicating the need for five desaturations in the substrate molecule. Subsequently, the product β-apo-120 -carotenal is converted to neurosporaxanthin by the aldehyde dehydrogenase YLO-1 in N. crassa [77] and by its ortholog CarD in F. fujikuroi [78]. 4. Plant CCDs The oxidative cleavage of carotenoids in plants leads to the production of a range of apocarotenoids compounds that serve critical functions including photoprotection, photosynthesis, pigmentation, and signaling [79,80]. Plant CCDs constitute the most abundant group identified so far. These CCDs have been classified into two large families based on whether they are involved or not in the production of abscisic acid (ABA), a hormone involved in drought stress responses and in bad and seed dormancy [81]. Those involved in ABA production are the nine-cis-epoxy-carotenoid-dioxygenases (NCEDs), which cleave 9-cis-violaxanthin and 9-cis-neoxanthin to xanthoxin, the precursor of ABA. In fact, the first CCD cloned from any organism was the Vp14 gene from maize that catalyzed the first committed step in ABA biosynthesis [81]. After the identification of Vp14, other CCD enzymes were isolated, including β-carotene oxygenase (BCO) in mammals involved in the synthesis of vitamin A, CCDs from microorganisms (mentioned in former sections), and other plant CCDs. In plants, five CCD groups have been identified so far–CCD1,

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CCD2, CCD4, CCD7, and CCD8. However, transcriptome and large-scale genomic sequencing projects in many different plant species are allowing the identification of new species-specific CCDs with yet unknown functions. In fact, biochemical studies have been reported for a limited number of plant CCDs, and the roles they play in different organisms are not fully understood. 4.1. CCD1 and CCD2 Subfamilies AtCCD1 from A. thaliana was the first carotenoid cleavage dioxygenase isolated not involved in ABA biosynthesis (Figure 1) [82]. The first cleavage activity reported for CCD1 enzymes was on the C9–C10 (C90 –C100 ) double bonds in C40 carotenoids, producing a colored C14 dialdehyde and two scent C13 products [82]. Homologues of this enzyme have been identified in many other plant species [83–91], and the studies performed on these CCD1 enzymes suggested additional cleavage activities on acyclic, monocyclic, and bicyclic carotenoids, such as ζ-carotene, lycopene, phytofluene, β-carotene, δ-carotene, zeaxanthin, lutein, and violaxanthin, and on different apocarotenoids, catalyzing the symmetric cleavage at C9–C10 (C90 –C100 ), C5–C6 (C50 –C60 ), C7–C8 (C70 –C80 ), and C13–C14 (C130 –C140 ) (Figure 3) [85,92–95]. The enzyme cleaved symmetrically at C9–C10 (C90 –C100 ) of acyclic and cyclic trans-carotenoids and did not cleave adjacently to a -cis double bond or an allenic bond found in some carotenoids [82]. Therefore, an asymmetric cleavage is observed in such cases. The C5–C6 or C50 –C60 activity of CCD1 enzymes on lycopene (or both), leading to the formation of the C8 ketone 6-methyl-5-hepten-2-one (MHO), was reported for the first time in tomato, maize, andA. thaliana [92] and was later detected in Cucumis melo [84], Rosa damascena [96], Oryza sativa [97], and Vitis vinifera [98]. The cleavage of C7–C8 and C7 0 –C80 double bonds of linear and monocyclic carotenoids constitutes a novel recognition site for the CCD1 plant subfamily. So far only detected by in vitro assays for the rice CCD1 enzyme [85], this activity allowed for the formation of C10 -aldehyde geranial, suggesting an alternative pathway for the geranial formation in plants. 4.1.1. Mode of Action and Functional Implications Initially, the CCD1 enzymes were suggested to be involved in the biosynthesis of apocarotenoid volatiles, such as geranylacetone, pseudoionone, and β-ionone [79], which possess an extremely low threshold for human perception [99]. Lowering of CCD1 expression in tomato or petunia led to significant reductions in the emission rates of β-ionone and geranylacetone, but did not lead to significant changes in the carotenoid concentration [90], suggesting additional roles for the CCD1 enzymes. In addition, the expression of CCD1 during tomato, strawberry, melon, and grape development, or in saffron flowers, did not mirror the emission of apocarotenoid volatiles [83,84,86,89,90]. Furthermore, experiments conducted to identify loci affecting volatile composition in tomato did not reveal the implication of CCD1 in the emissions of apocarotenoid volatiles [100]. These are produced from C40 carotenoids localized in plastids, and the CCD1 enzymes lack plastid targeting signals and are cytoplasmatic [87,101], suggesting that CCD1 enzymes mainly act in planta as scavengers of carotenoid degradation products of different chain lengths rather than primary cleavers of intact carotenoids [80,85,93,94,98,102]. The key roles of carotenoids in photosynthesis, photomorphogenesis, and plant development suggest that their biosynthesis and degradation is coordinately regulated with processes such as plastid biogenesis, flowering, and fruit development [103]. In addition, the link of carotenoid biosynthesis with those of gibberellin, ABA, and strigolactone phytohormones implies that changes in the composition or content of carotenoids might bring about physiological or biochemical shiftings in plants [4]. In 2008, a role of CCD1 in the production of strigolactones (SLs) during the arbuscular mycorrhiza symbiosis in roots was described [94]. CCD1 cleaves the C27 apocarotenoid derivatives produced by the activity of CCD7, which is also involved in the biosynthesis of SLs [104], producing C13 α-ionol and C14 mycorradicin, which are indicators of a well-established and functional symbiosis [105], reducing SL production and avoiding over-colonization [106].

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CCD1 could be also important in plant stress responses. Enhanced CCD1 expression during berry development has been associated with the increased osmotic stress that occurs during ripening, resulting in leaky membranes and concomitant chloroplast degradation [98]. In addition, CCD1 expression is induced during leaf senescence [89,107], suggesting that the enzyme-mediated degradation of carotenoids, apocarotenoids, or both is catalyzed by cytosolic CCD1 enzymes. Furthermore, plant–insect interactions, extreme temperatures, high irradiance, or ultraviolet (UV) stress Int. the J. Mol. Sci. 2016, 17, 1781 of 37 induce production of β-ionone and β-cyclocitral among other apocarotenoid volatiles15[108,109]. Int. J. Mol. Sci. 2016, 17, 1781 15 of 37 CCD1 expression has been detected in all tested tissues in different plant species [89–91,95,98,110,111], CCD1 expression has been detected in all tested tissues in different plant species [89–91,95,98,110,111], but its expression is stimulated abiotic or tissues ABA treatment CCD1 expression has been by detected in stress all tested in different[112,113]. plant species [89–91,95,98,110,111], but its expression is stimulated by abiotic stress or ABA treatment [112,113]. but its expression is stimulated by abiotic stress or ABA treatment [112,113].

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ble 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases.

e-oxygenase is PCC 7806

NosCCD (NSC1) Nostoc sp. PCC 7120 β-ionone ( ) + Apo-8,10′ -apocarotene-dial ( ) Other minority products ( , ) β-ionone ( ) + Apo-10,10′ -apocarotene-dial ( )

Enzymes and Products MtCCO Mycobacterium NSC3 Nostoc sp. PCC 7120 tuberculosis β-apo-13-carotenone β-apo-13-carotenone ( ) ( ) β-apo-14′-carotenal Retinal ( ) Transretinal ( )

β-apo-13carotenone ( )

SaCCO Sphingopyxis alaskensis Apo-12′carotenal ( ) Apo-10′carotenal ( )

PpCCO Plesiocystis pacifica

No (in vitro)

No

β-apo-13-carotenone ( ) Retinal (

)

3-OH-β-ionone ( ) + Apo8,10′-apocarotene -dial ( )

3-OH-β-apo-13-carotenone ( ) 3-OH-retinal ( )

3-OH-β-ionone ( ) + Apo10,10′-apocarotene -dial ( )

3-OH-β-apo-13-carotenone ( ) 3-OH-retinal ( )

β-ionone ( ) + Apo-8,10′ -apocarotene-dial ( )

β-apo-13carotenone ( )

citral ( , ) dial ( , )

Apo-10,10′-apocarotene -dial ( )

β-apo-13-carotenone ( ) Retinal ( ) β-apo-14′-carotenal ( )

droxi-βocitral , ) dial ( ,

3-OH-β-ionone ( ) Apo-10,10′-apocarotene -dial ( )

3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-14′-carotenal ( ) 3-OH-retinal ( ) 3-OH-β-apo-11-carotenal ?

)

NACOX1 Novosphingobium

No

No

Int. J. Mol. No Sci. 2016, 17, 1781 No

No (in vitro)

Apo-13′zeaxanthinone ( ) Apo-14′zeaxanthinal ( ) of37 37 99of 9 of 37 Apo-12′zeaxanthinal ( )

Apo-12′Apo-12′Yes (in Apo-12′vivo) 3-OH-β-ionone ( ) poorly cantaxanthinalUnknown cantaxanthinal (( )) cantaxanthinal product ( ) 3-OH, 4-oxo-β-inone ( ) 3-OH-β-ionone(( )) 3-OH-β-ionone ( Figure Apo-13′3-OH-β-ionone Apo-13′) 3-OH-β-apo-13-carotenone Apo-13′( ) 3. Activity of plant CCD1 and CCD2 subfamilies, showing their divergence in substrate Apo-10,10′-apocarotene 3-OH,4-oxo-β-inone 4-oxo-β-inone3-OH, astaxanthinone Figure Activity of plant CCD1CCD1 and and CCD2 subfamilies, showing their divergence in substrate 3-OH, (( )) 4-oxo-β-inone astaxanthinone (( )) astaxanthinone ( 3-OH-β-apo-14′-carotenal )3. ( ) Figure 3. Activity CCD2 subfamilies, showing their divergence ( of) plant -dial (in ) substrate specificity and cleavage sites on the following substrates: (A) ζ-carotene and lycopene; (B) β-carotene; Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene Apo-14′Apo-10,10′-apocarotene Apo-14′3-OH-retinal ( ) No specificity and cleavage sites on the following substrates: (A) ζ-carotene lycopene; β-carotene; References [19] and [30,33] specificity and cleavage sites on the following substrates: (A) ζ-carotene and lycopene; (B) β-carotene; -dial(( )) astaxanthinal -dial astaxanthinal )) astaxanthinal and3-OH-α-apo-15′-carotenal (C) zeaxantine. Symbols indicate double bonds where oxygenases cleave. (:( C5–C6; : C7–C8; -dial ( ) ( ) Symbols indicate double where[38]oxygenases cleave. C7–C8; and (C) and zeaxantine. indicate bonds oxygenases cleave. : C5–C6; [30,33] [36]zeaxantine. [31]Symbols [37]double [38]bonds [38] and (C) Symbols indicate double bonds where oxygenases cleave. C5–C6; C7–C8; [36] [31] [37] [38] [38] [19] [30,33] [30,33] [36] [31] [37] [38] : :C7–C8; : C9–C10 : C13–C14; : C13′–C14′; :where C11′–C12′. ( ) C9′–C10′; No

Astaxanthin

No

[36]

: C9–C10; : C13–C14; 0and 0 ; :: C15–C15′; 0:–C14 0 ; enzyme 0 –C12 0 . at : C7′–C8′. When an cleaves different the predominant product is indicated in b : and C9–C10 C9′–C10′; : C13–C14; C13′–C14′; : C11′–C12′. cleave. C7–C8; C9–C10; C13–C14; C15–C15′; C13′–C14′; C11′-C12′; C9′–C10′;positions, 4-oxo-β-ionone ( ::) C9–C10; nases cleave. :: C7–C8; :: C13–C14; C13′–C14′; :: C11′-C12′; :: C9′–C10′; snases where oxygenases cleave. : C7–C8; :C9 C9–C10; C13–C14; C15–C15′; C13′–C14′; C11′-C12′; : C9′–C10′; C9–C10 –C10 C13–C14; :: C13 C11 Apo-10,10′-apocarotene not available. taves positions, the predominant product is indicated indicated in bold. bold. Lack ofin bold means no product preference orinformation information positions, the predominant product is in Lack of bold means preference or at different positions, the predominant product is indicated bold. Lackno ofproduct bold means no product preference or information No 4.1.2. CCD1 Gene Family and Genomic Organization -dial ( ) 4.1.2. β-ionone ( )

4.1.2.Gene CCD1Family Gene Family and Genomic Organization CCD1 and Genomic Organization

The CCD1 family is present in some plant species as a multigene family (Table S1), and studies

The CCD1 family is present in some speciesvarying as a multigene familyApo-13′-canta(Tableand S1), different and studies on tomato CCD1 enzymes haveplant suggested expression 2 x 4-oxo-β-iononeThe ( ) maize CCD1and family is present in some plant species as a multigene family profiles (Table S1), and studies on xanthinone ( )and different on maize and tomato CCD1 enzymes have suggested varying expression profiles Apo-10,10′-apocarotene inCCD1 their activity different varying substrates and in their double bond preferences Apo-14′maizespecificities and tomato enzymestowards have suggested expression profiles and different specificities -dial ( ) specificities in In their activity towards species, differentthere substrates in encoding their double bond preferences cantaxanthinal ( )enzymes, [90,92,93,102,114]. most of the analyzed are twoand genes for CCD1

in their activity towards different substrates and in their double mostare ofpresent the analyzed species, there arechromosome twobond genespreferences encoding for[90,92,93,102,114]. CCD1 enzymes, and[90,92,93,102,114]. in many cases the In genes in tandem in the same (Table S1). The identities In most of the analyzed species, there are two genes encoding for CCD1 enzymes, andidentities in many and in many cases the genes are present in tandem in the same chromosome (Table S1). The among the paralogous CCD1 genes range between 90% and 99%. All the CCD1 genes show several among the paralogous CCD1 genes range between 90% and[115]. 99%.Moreover, All the CCD1 genes show several introns in their sequence [115], these positions being conserved the presence of such introns in their sequence [115], these positions being conserved [115]. Moreover, the presence such introns allows for the generation of different splice variants (Table S1). These variants may differofas introns allows for the generation of different splice variants (Table S1). These variants may differ well in the untranslated regions, with implications in transcript stability, localization, translation, or as well in the untranslated regions, implications in transcript stability, localization, translation, a combination thereof. It is likely thatwith the truncated proteins from the CCD1 mRNA isoforms may act or a combination thereof. It is likely that the truncated proteins from the CCD1 mRNA isoforms may act

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cases the genes are present in tandem in the same chromosome (Table S1). The identities among the paralogous CCD1 genes range between 90% and 99%. All the CCD1 genes show several introns in their sequence [115], these positions being conserved [115]. Moreover, the presence of such introns allows for the generation of different splice variants (Table S1). These variants may differ as well in the untranslated regions, with implications in transcript stability, localization, translation, or a combination thereof. It is likely that the truncated proteins from the CCD1 mRNA isoforms may act as dominant-negative regulators. In fact, several reports provide evidences of a dominant regulatory role for truncated proteins generated by splice variants in plants [116]. AtCCD1 has been suggested to act as a dimer [82], and the presence of these variants could be part of a regulatory mechanism for CCD1 activity. The phylogenetic analysis of the CCD1 proteins reveals several main clusters that fit with the phylogenetic relationships in embryophytes (Figure S1). In the angiosperm group, there are three sub-clusters corresponding to monocots, dicots (excluding crucifers), and crucifers. The latter sub-cluster of Brassicaceae species is more related to the monocot group than to the dicot group, suggesting a specific function for CCD1 in these plants. 4.1.3. Mode of Action of CCD2 Recently, a close CCD subfamily related to the CCD1 family has been identified in Crocus species [117,118]. This subfamily, named as CCD2 (Figure 1), is involved in the formation of the apocarotenoid crocetin, which accumulates in stigma tissue at high levels [119]. Crocetin is derived by bio-oxidative cleavage of zeaxanthin by a C7–C8 and C70 –C80 cleavage reaction catalyzed by CsCCD2 [117]. The expression of CsCCD2 is restricted to the stigma in saffron, and it mirrors the levels of crocetin in this tissue during its development [89,117]. The same behavior is observed for CaCCD2, a CCD2 homologue isolated from the spring crocus C. ancyrensis [118]. Both CCD2 enzymes are plastidic, a major difference with the CCD1 subfamily [118]. No other CCD2 homologues have been identified in other organisms, presumably due to the uncommon ability to synthesize crocetin in plants [120] or bacteria [121]. Besides zeaxanthin, CCD2 is able to recognize and cleave lutein and 3-OH-β-apocarotenals at the C7–C8 position, but it does not cleave β-carotene, lycopene, or β-cryptoxanthin [117]. 4.1.4. Structure of CCD1 and CCD2 Enzymes Based on the crystal structure of VP14 [122], structural models have been built for CCD1 and CCD2. VP14 is a globular protein that showed a characteristic hydrophobic patch formed by the antiparallel α-helices presumably involved in membrane interaction [28]. CCD1 and CCD2 enzymes display from 58% to 39% overall identity with VP14. Specially conserved are the structural elements of the CCD enzymes. Three regions have been identified in VP14, implicated in the recognition of the bond to be cleaved [122]. The first one is Val478, which is a substitute for a Phe residue in CCD1 and CCD2 enzymes. This substitution is present in many others CCDs. The second region is a loop formed by residues 499 to 503 in VP14. This loop is not present in CCD1 and CCD2 enzymes, in which conserved Leu170 in Vp14 was replaced by Trp. In Vp14, the loop and the leucine residue form a pocket where the second ring of the carotenoid accommodates in such a way that the C11–C12 double bond of the substrate should be close to the Fe2+ . The third region, located in the active center, comprises three Phe residues in Vp14 (Phe171, Phe411, and Phe589) and is conserved in all NCED and CCD1 enzymes; however, in CCD2, only one Phe residue is conserved. 4.1.5. Regulation of CCD1 and CCD2 Enzymes As stated before, CsCCD2 expression is regulated during the development of the stigma and the enzyme is localized in chromoplasts [123] where catalyzes crocetin biosynthesis. Crocetin is oxidated and glucosylated thereafter [124], generating crocins that accumulate in the vacuole. The mechanisms involved in CsCCD2 expression and activity have recently been elucidated [123].

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In this study, the authors showed that high temperatures repressed CsCCD2 expression, while low temperatures have an opposite effect by upregulating its expression. In saffron plants, the highest expression levels of CsCCD2 are related to the lowest temperatures. Several classes of cis-regulatory elements were identified in the promoter of CsCCD2. The regulation of carotenoid biosynthesis by light and dark conditions has been investigated in red pepper [125], in tomato leaves [126], and in citrus, where the expression levels of the carotenogenic genes are affected by circadian rhythms [127,128]. Additionally, a new mechanism of regulation by alternative splicing has been observed for CsCCD2, [123]. The obtained data agree with the predicted function of alternative splicing in genes with a circadian clock expression pattern, where oscillations of protein expression levels require rapid adjustments in mRNA levels over the course of a day [129]. 4.2. The CCD4 Subfamily The CCD4 subfamily is present in all flowering plants, usually composed by several members (Figure 1) [115,130]. Their functions have been directly linked to coloration and production of aroma volatiles in flower and fruit tissues [89,98,131–133]. More recently, CCD4 activity has been reported in seeds, leaves, and roots associated with carotenoid turnover [107,134] or with the production of novel apocarotenoid-derived signals [135]. The first member of this subfamily was identified in A. thaliana [18]; however, its role in carotenoid catabolism was initially described for CCD4a from Chrysantemum morifolium, whose activity is responsible for the absence of carotenoid accumulation in petals of white flowers [136]. All CCD4 proteins contain a plastid target peptide; moreover, suborganellar studies in different plant species localized these enzymes in the plastoglobuli together with other carotenoid biosynthetic enzymes, which allows direct access to their substrates [131,137–141]. In both chloro- and chromoplasts, CCD4 is one of the major components of plastoglobuli proteomes [ 137,140], indicating a role for this enzyme in the regulation of carotenoid metabolism in plastid types with marked differences in functionality and carotenoid composition [142]. Moreover, CCD4 protein level is strongly reduced in Arabidopsis mutants defective in the ABC1K1 and ABC1K3 plastoglobuli kinases, suggesting phosphorylation as a post-transcriptional modification necessary for CCD4 protein stability and supporting main activity and localization in plastoglobuli [143]. 4.2.1. Enzyme Structure and Functional Implications As found for other CCDs, molecular modeling of CCD4 enzymes from the structure of the 9-cis-epoxycarotenoid dioxygenase from maize (VP14/NCED) revealed a well-conserved structure among the CCD family [130,133]. The analyzed CCD4s display two main functional domains: the helical domain composed of two antiparallel α-helices, which might penetrate in the hydrophobic core of the plastoglobule where carotenoids accumulate [130,138], and the β-propeller structure that forms a long central tunnel, where Fe2+ is coordinated with four conserved His in the active center [130,133]. However, other critical amino acids for NCED activity are not always conserved among all CCD4 members [130,133]. As was the case in CCD1 and CCD2 enzymes, a large number of CCD4 proteins display a Phe residue instead of the conserved Val478 found in NCED, with the exception of CCD4 from alfalfa (CCD4a and b), grape (CCD4e), potato (CCD4b), poplar (CCD4c), and citrus (CCD4b1), where it is substituted by Leu; however, in citrus CCD4c, Val478 is replaced by Met. Other critical residues in VP14 are three Phe at positions 171, 411, and 589, from which only Phe589 is conserved in all CCD4, while Phe411 is highly variable, and Phe171, which also takes part of the motif FDG, is partially conserved and usually substituted by Leu. Another important region for VP14 activity is a loop located on the back side of the substrate pocket, involving residues 499–503 and Leu170. In contrast with CCD1 and CCD2, residues in this loop are highly conserved in the CCD4 subfamily; however, the citrus CCD4b1 is a relevant exception, with the Glu499 and Pro500 reported in VP14 substituted by Lys and Glu, respectively. The Leu170 in VP14 is also well conserved in all CCD4s; however, in CCD4b1, CCD4b, and CCD4e from citrus, potato, and poplar, it is replaced by

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Phe and in citrus CCD4c by Pro. Although it is difficult to determine exactly the impact of these residue changes in the enzyme activity, it is likely that they may modulate the carotenoid substrate specificity, the cleavage position, or both. Interestingly, functional assays performed with citrus CCD4b1, which accumulates several non-conservative amino acid substitutions, show an unusual cleavage position, having as preferential substrates the hydroxylated β-ionone ring of the xanthophylls. Future studies on comparative 3D modeling of the different members of this subfamily will provide clues as to substrate specificity or cleavage position in carotenoid backbone, which may help to understand their role in vivo. 4.2.2. Gene Family and Genomic Organization The number of genes for CCD4 enzymes is very variable among different species, with at least two genes identified in most plants [115,144]. The presence of one single CCD4 gene has been reported only in a limited number of plant species with an available genome sequence, such as A. thaliana, Carica papaya (papaya), Prunus persica (peach), and Sorghum bicolor (sorghum) [115,130,144]. By contrast, the largest number of CCD4 genes has been found inCitrus sp. (a, b1, b2, c and d), Populus trichocarpa (a–e), and Chrysanthemum morifolium (a-1–a-4 and b), with five members and with at least one of them being a putative pseudogene [115,133,145,146]. Four members have been identified in Vitis vinifera (grapevine) (a, b, c, and e) or Brassica napus (rapeseed) (A1, A8, C1, and C3), while three are present in Theobroma cacao (cacao) [98,130]. Another common genomic feature of CCD4 genes is the low presence of introns, most of them being intron-less or containing a single intron [ 115,144]. Moreover, intron size and position are quite variable among different species. A comprehensive comparative analysis of the genomic structure of CCD4 from diverse plants showed that introns are located in three different sites and only those located at the 30 end of the gene maintain a conserved position [115]. Exceptionally, only a few CCD4 genes contain more than one intron, such as Osmathus fragans CCD4 and Solanum lycopersicum CCD4a and b, each with two introns, Citrus sp. CCD4d with two or four introns, and an uncharacterized CCD4 from apple (Malus domestica) with 85% identity at the protein level to apple CCD4a [96] with six introns. The degree of protein sequence identity among CCD4 members is quite variable, ranging from 30% to 98%, with typical values around 50%–70% [115,130,133,147,148]. In multigene CCD4 families, a high degree of homology (85%–98% identity at the protein level) is usually found between the different members, frequently located in tandem on the same chromosome (i.e., tomato CCD4a and b, citrus CCD4b1 and b2, chrysamthemum CCD4a-1 to a-4, or Populus CCD4c to e) [115,133]. As expected, the highest variability is found at the plastid target peptide sequences, while a higher conservation is found in the motifs described as essential for CCD activity [6,28,149]. The moderate conservation within the different CCD4 members of the same family (i.e., citrus CCD4b1 versus CCD4c or grapevine CCD4a/b versus CCD4c/e), and the location in different clusters in the phylogenetic analysis (Figure S2) suggest a divergent evolution and functionality of these members. 4.2.3. CCD4 Activity: Carotenoid Substrates and Cleavage Products In contrast to other CCD subfamilies, such as NCED or CCD7/8, functional assays of CCD4 indicate that the members are rather heterogeneous in respect of carotenoid substrates and cleavage positions (Table 4). The functional characterization of CCD4 has been an active field of research in the last decade by following three basic strategies: (i) in vivo assays of CCD4-defective plant mutants or CCD4-overexpressing or repressing transgenic plants; (ii) in vivo assays of bacteria over-accumulating different carotenoids and co-expressing CCD4; and (iii) in vitro enzymatic assays performed with recombinant CCD4. Table 4 summarizes the information currently available on CCD4 enzyme activity. The first comparative study of substrate preferences and cleavage positions of CCD4 enzymes was obtained from in vitro and in vivo assays performed with five CCD4s isolated from Arabidopsis, rose (R. damascene), osmanthus (O. fragans), apple (M. domestica), and chrysanthemum (C. morifolium) [96]. Most of these CCD4 cleaved β-carotene at C9–C10 and C90 –C100 positions,

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rendering C13 β-ionone, while no activity was detected on hydroxylated xanthophylls [96]. Further research using Arabidopsis ccd4 mutants or carotenoid over-accumulating lines revealed that epoxy-xanthophylls (mainly violaxanthin) are likely the substrates in Arabidopsis leaves [107,134] to render C13 apocarotenoids (cleavage at C9–C10 or C90 –C100 double bonds) that undergo further glycosylation [134]. Interestingly, changes in carotenoid composition and apocarotenoid profile in ccd4 Arabidopsis mutants, during seed maturation or in roots of hyperaccumulating β-carotene plants, suggest that CCD4 also accepts β-carotene as substrate [107,134]. One of the two CCD4s from potato, in this work designated as CCD4a (Figure S2), was functionally characterized. Analysis of the carotenoid profile in tubers from RNAi CCD4 potato lines suggested violaxanthin as the main substrate [150]. However, in-depth biochemical characterization of potato CCD4a using in vitro and in vivo assays points to β-carotene as its main substrate, cleaved at C9–C10 or C90 –C100 positions [148]. In saffron, in vivo assays showed that CCD4a and b cleave β-carotene and zeaxanthin most likely at the C9–C10 and C90 –C100 double bonds, while CCD4c cleaves other xanthophylls such as lutein [89,130,151]. In vivo assays of grapevine CCD4a and b indicate that these enzymes, in contrast to all CCD4s characterized so far, do not cleave cyclic carotenoids, with the exception of β-carotene, cleaving linear carotenes neurosporene, lycopene, and β-carotene—only CCD4b preferentially at C9–C10 and C9 0 –100 positions [98]. The carotenoid profile from rape (Brassica napus) flowers suggests that the member CCD4_C3 cleaves α-carotene or δ-carotene at the C9–C10 position to render α-ionone [152]. Interestingly, in vitro and in vivo assays show that Citrus sp. CCD4b1/CCD4 accepts β-ring hydroxylated xanthophylls as preferential substrates, but also accepts β-carotene or α-carotene. In contrast to the other plant CCD4s, citrus CCD4b1 and CCD4 cleaves the carotenoid backbone at C7–C8 or C70 –C80 double bonds, resembling CCD2 activity, but only at one side of the molecule [131,133]. In summary, in vitro and in vivo assays for most of the CCD4 enzymes point to β-carotene and a C9–C10 or C90 –C100 double bond cleavage as a favorite substrate and positions, respectively, although some relevant exceptions, such as citrus or grape CCD4, have been identified regarding the substrate or cleavage site. In particular, the exclusive cleavage activity reported for citrus CCD4b1 and CCD4, together with its restricted expression pattern, may indicate that this enzyme belongs to a novel class of CCD more functionally related to the recently characterized CCD2 from saffron [117,118]. In planta data, derived from the analysis of apocarotenoid profiles, the alteration in carotenoid complement in ccd4 mutants and overexpressed or repressed CCD4 plants, or both, point to xanthophylls as the preferential substrates in vivo [130,133,134,145]. Nevertheless, in some species and tissues, such as potato tubers, chrysanthemum petals, and peach fruit, β-carotene cannot be ruled out as the primary target since alterations in the xanthophylls composition in defective or overexpressing CCD4 plants can be explained by a reduction or increase in β-carotene content as the precursor of β-xanthophylls [136,150,153]. These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the types of assay. In the in vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98].

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Table 4. Functional characterization of CCD4 enzymes. Species

Arabidopsis thaliana

Protein

CCD4

Assay a

Parent Carotenoid

Cleavage Position

Product Detected

References

in planta

β-carotene, Violaxanthin

n.d.

n.d.

[107]

in planta

Phytofluene, ζ-carotene

n.d.

n.d.

[135]

C9–C10 or

C90 –C100

in planta (leaf)

Epoxy-β-xanthophylls

C13 -glycosids

[134]

in planta (root)

β-carotene

n.d.

long-chain free apocarotenals (C15 to C30 )

[134]

in vitro

Apo-β-caroten-80 -al

C9–C10

β-ionone

[96]

Brassica napus

CCD4_C3

in planta

α-carotene, δ-carotene

C9–C10

α-ionone

[152]

Chrysantemum morifolium

CCD4a

in vivo and in vitro

β-carotene

C9–C10 (C90 –C100 )

β-ionone

[96]

in vitro

β-carotene, β-cryptoxanthin, Zeaxanthin, Lutein, α-carotene

C7–C8 or C70 –C80

3-OH-apo-β-8-carotenal, apo-β-8-carotenal (C30 ); β-cyclocitral and 3-OH-β-cyclocitral (C10 )

[133]

3-OH-apo-β-8-carotenal

[131]

β-ionone, β-OH-ionone

[89,147]

β-ionone, β-cyclocitral

[130]

Citrus clementina Citrus unshiu

CCD4b1 CCD4 CCD4a/b

in vitro and in vivo in vivo in vivo

Crocus sativus CCD4c

Malus domestica Osmanthus fragans Rosa damascena

Solanum tuberosum

Vitis vinifera

a

CCD4 CCD4 CCD4

CCD4

β-cryptoxanthin, Zeaxanthin β-carotene, Zeaxanthin β-carotene

in planta

C7–C8 or C9–C10

C70 –C80

(C90 –C100 )

C9–C10 or C90 –C100 C7–C8 or C70 –C80

in planta

Lutein

C9–C10

Megastigma-4,6,8-triene (derived from 3-OH-α-ionone)

[130]

in vivo

β-carotene

C9–C10 (C90 –C100 )

β-ionone

[96]

(C90 –C100 )

in vivo

β-carotene

C9–C10

β-ionone

[96]

in vivo

β-carotene

C9–C10 (C90 –C100 )

β-ionone

[96]

in vitro

apo-β-8-carotenal

C9–C10

β-ionone

[96]

in planta

Violaxanthin

n.d.

n.d.

[150]

in vitro and in vivo

β-carotene

C9–C10 or C90 –C100

Apo-β-caroten-100 -al; β-ionone

[153]

in vitro

α-carotene, Lutein, Zeaxanthin, β-cryptoxanthin

C9–C10 or C90 –C100

3-OH-β-apo-100 -carotenal, β-apo-100 -carotenal3-OH-ε-apo-10´-carotenal

[153]

α-ionone,

[98]

Geranylacetone

[98]

CCD4a/b

in vivo

ε-carotene,

CCD4a/b

in vivo

Neurosporene

CCD4a/b

in vivo

Lycopene

CCD4b

in vivo

ζ-carotene

C9–C10

(C90 –C100 )

C90 –C100 C5–C6

(C50 –C60 )

C9–C10 (C90 –C100 )

6-methyl-5-hepten-2-one

[98]

α-ionone, Geranylacetone

[98]

In planta refers to data inferred from changes in carotenoid and/or apocarotenoid profiles in overexpressing or knockout CCD4 mutants; in vivo assay indicates data obtained from bacteria over-accumulating carotenoids and expressing CCD4; in vitro assay refers to enzymatic assays performed with recombinant CCD4 enzyme. n.d. not determined.

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These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the types of assay. In the in vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98]. In summary, in vitro and in vivo assays for most of the CCD4 enzymes point to β-carotene and C9–C10 or C90 –C100 double bond cleavage as the favorite substrate and positions, respectively, although some relevant exceptions, such as citrus or grape CCD4, have been identified regarding the substrate or cleavage site. In particular, the exclusive cleavage activity reported for citrus CCD4b1 and CCD4, together with its restricted expression pattern, may indicate that this enzyme belongs to a novel class of CCD more functionally related to the recently characterized CCD2 from saffron [117,118]. In planta data, derived from the analysis of apocarotenoid profiles, the alteration in carotenoid complement in ccd4 mutants and overexpressed/repressed CCD4 plants, or both, point to xanthophylls as the preferential substrates in vivo [130,133,134,145]. Nevertheless, in some species and tissues such as potato tubers, chrysanthemum petals, or peach fruit, β-carotene cannot be ruled out as the primary target since alterations in the xanthophylls composition in defective or overexpressing CCD4 plants can be explained by a reduction or increase in β-carotene content as the precursor of β-xanthophylls [136,150,153]. These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the type of assay. In thein vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98]. 4.2.4. Expression Pattern in Plant Tissues CCD4 genes are predominantly expressed in flower and fruit tissues, suggesting specific roles in these organs [130,147]. Usually at least one CCD4 member is highly or exclusively expressed in flowers, and its function has been related to the production of nor-isoprenoid aroma volatiles to attract pollinators [96,133,146,148,154,155]. In chrysantemum, Osmanthus, and cabbage, the expression of a specific CCD4 gene in petals is responsible for carotenoid degradation and a lack of flower coloration [136,145,152]. In chrysanthemum, CCD4a is represented by a small multigene family, and the white to yellow color gradation in petals among different cultivars can be explained by different combinations and levels of expression of specific CCD4a genes [145]. In the flower petals of lily (Lilium brownii var. colchesteri), CCD4 transcript levels are associated with a loss of carotenoid content during anthesis, resulting in flower whitening [ 156]. In saffron, the expression of CCD4a/b correlates with the production of the nor-isoprenoid β-ionone in flower tissues, while CCD4c is restricted

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to stigmas and shows a highly regulated expression pattern during flower development [89,130]. In other species, such as sweet orange, tomato, and potato, at least one CCD4 gene is expressed in flowers, but their involvement in carotenoid degradation has not been elucidated [133,148,150]. Expression of CCD4 genes has also been reported in many fruits and its role has been associated with the biosynthesis of nor-isoprenoid volatiles or apocarotenoids pigments, which may attract animals to facilitate seed dispersion. In grape, CCD4a and b genes are expressed in the berry, but only CCD4b shows a fruit-specific expression profile, highly upregulated during ripening, linking its activity to nor-isoprenoid volatiles production [98]. In summer squash (Cucurbita pepo) varieties with white, green, and yellow-orange coloration, the expression of two CCD4 genes (a, b) correlates inversely with carotenoid accumulation in the peel and flesh; therefore, both genes are good candidates for regulating fruit color [111,157]. One of the best-characterized examples of the CCD4 role in fruit pigmentation has been described in peach (Prunus persica). White-fleshed peach cultivars contain at least 10 times less carotenoid concentration and higher nor-isoprenoid carotenoid-derived volatiles than the yellow ones [158]. Recently, several studies have established that alterations in the CCD4 gene are directly related to carotenoid content in yellow peach varieties. In yellow cultivars, CCD4 gene expression is significantly reduced, the formation of a truncated CCD4 protein avoids carotenoids degradation in fruit mesocarp, or both [159–162]. In mandarins and oranges, the specific expression of the CCD4b1/CCD4 gene in the fruit peel during ripening is responsible for the asymmetric cleavage of β-cryptoxanthin and zeaxanthin to generate the C30 apocarotenoids 3-OH-β-8-apocarotenal (β-citraurin) and β-apo-8-apocarotenal [131,133], respectively. It is interesting to note that, due to the intense orange-red color of these C30 apocarotenoids, the citrus CCD4 activity enhances fruit pigmentation, in contrast to the other CCD4s described so far whose activity is associated with color loss. In other species, such as bitter melon, goji berry, and tomato, the expression of at least one CCD4 gene has been reported in ripened fruits, but no relationship has been established with carotenoid content [148,155,163]. In potato tubers, compared with the yellow ones, CCD4 activity regulates coloration, as indicated by the elevated CCD4 expression in mature white-fleshed tubers [150]. Moreover, in CCD4 RNAi potato lines, total carotenoid content was enhanced in tubers (up to 5.6-fold) and flowers, but not in other plant organs [150]. The function of this enzyme in tubers is probably associated with stress response since the potato tubers from RNAi lines showed diverse developmental alterations linked to heat stress phenotypes [150]. Recently, it has been proposed that an apocarotenoid derived from β-carotene in potato tubers could act as a signaling molecule in stress responses [153]. In other plant species, CCD4 expression is also modulated by specific abiotic stresses. Saffron CCD4a and b are expressed in leaf tissues upon dehydration or heat stress as well as in senescence leaves [89]. Moreover, CCD4c is upregulated in flower stigmas subjected to different stress treatments, such as osmotic, wounding, and cold or heat stresses [130]. In B. rapa and B. oleracea seedlings, CCD4 transcript levels increase in response to different abiotic stresses as well as to exogenous treatments with SLs and ABA phytohormones, suggesting their involvement in plant stress resistance response [152]. In other processes involving cellular responses to stress, such as dark-induced leaf senescence or seed maturation and desiccation, a major role of CCD4 in carotenoid degradation has been proposed [107]. These processes are associated with severe alterations in chloroplast structures, causing disassembly of photosystems and light-harvesting complexes and the release of carotenoids, in which CCD4 activity may play a crucial role in modulating turnover of β,β-carotenoids. By contrast, high-light stress in Arabidopsis leaves causes a strong downregulation in CCD4 expression [164], which may prevent cleavage of xanthophylls with a photoprotective function [134]. A role of CCD4 maintaining carotenoid homeostasis has also been reported in Arabidopsis overexpressing PSY [134]. In these plants, CCD4 activity is essential for the formation of apocarotenoid (C13 ) glycosides (derived from epoxidized xanthophylls) in leaves and long-chain apocarotenoids (derived from β-carotene) in roots. Moreover, a lethal phenotype was observed in PSY-overexpressing seedlings defective in CCD4 activity, supporting the key role of CCD4 as a first step in detoxifying excess of carotenoids

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into apocarotenoid glycosides [134]. The CCD4 activity is also involved in the formation of an uncharacterized apocarotenoid, most likely derived from ζ-carotene, phytofluene, or both, participating in a novel signaling process that regulates early chloroplasts and leaf development [135]. In summary, the presence of different CCD4 members with tissue- and organ-specific expression patterns in many plant species indicates a high functional diversification within this subfamily. In the last few years, we have gained much knowledge on the physiological, biochemical, and molecular regulation of CCD4 enzymes and their presumed involvements in different processes. In one case, the function is concerned with the production of apocarotenoids in flowers and fruits to attract pollinators and seed dispersers, which has eco-physiological and reproductive implications. In another case, the role is directly involved in carotenoid turnover, related to specific stress conditions and alterations of the carotenoid pathway. A third function was recently identified with the involvement of CCD4 activity in the formation of an apocarotenoid-derived signal that regulates early chloroplasts and leaf development. Future research on different CCD4 members in multigene families, including structural, functional, and spatial studies as well potential interactions with other enzymes of the carotenoid pathway, will help towards a better understanding as to how carotenoid content is modulated in different plant organs. 4.3. The CCD7 and CCD8 Subfamilies The first report on CCD7 and CCD8 enzymes in plants derives from the max4 mutant of Arabidopsis, affected in the AtCCD8 gene (Figure 1). The mutants exhibit an increase in lateral branching, which reminiscent of the phenotype of the max3 mutant, which shows an increase in lateral branching as a result of a mutation in the AtCCD7 gene, indicating that both AtCCD8 and AtCCD7 are involved in this developmental process [165,166]. The biochemical characterization of AtCCD7 and AtCCD8 [167] demonstrated that AtCCD7 catalyzes a C9–C10 cleavage of β-carotene to produce 100 -apo-β-carotenal (C27 ) and β-ionone (C13 ). The AtCCD8 protein is able to catalyze a secondary cleavage of 100 -apo-β-carotenal at the C13–C14 position to produce 13-apo-β-carotenone (C18 ). However, it has been shown that CCD7 is also active on many other carotenoid substrates [166,167]. In contrast to CCD1, which has a cytoplasmatic location, CCD7 and CCD8 are localized in plastids, as CCD4 and CCD2 are, the predominant sites for carotenoid accumulation [87]. The CCD7 and CCD8 enzymes are involved in the biosynthesis of a relatively novel class of apocarotenoid hormones, the strigolactones (SLs), which control lateral shoot growth and which appear to be well conserved among the studied plant species. Branching mutants from Arabidopsis, petunia, pea, and rice lacking CCD7 or CCD8 have reduced SL concentrations, and applications of synthetic SLs to the mutant restores the wild-type branching phenotype [87,168,169]. In tomato, a reduction of CCD7 expression increases branching [170]. This is the case of the tomato mutant Sl-ORT1, which is deficient in SLs and has reduced CCD7 expression [171]. These enzymes have been identified in all high plant genomes sequenced up to date. SLs are derived from carotenoids, thus belonging to the class of the apocarotenoids that function as signaling molecules. This is exemplified by their role promoting germination of the parasitic plants Striga and Orobanche [172] or their involvement in symbiotic interaction with arbuscular mycorrhizal fungi [173]. CCD7 and CCD8 are involved in sequential cleavage reactions needed for the synthesis of SLs. These metabolites share a common C19 structure consisting of a tricyclic lactone connected via an enol ether bridge to a second lactone (Figure 4). The first steps of SL synthesis involve isomerization and dioxygenase-mediated cleavage of a carotenoid precursor via plastidic isomerase action by D27, followed by the cleavage activity of CCD7 and CCD8. MAX1 encodes a cytP450 enzyme predicted to act downstream of CCD7 and CCD8, and is required for the synthesis of active SLs. SL signaling requires the hormone-dependent interaction of the α/β hydrolase protein DWARF 14 (D14), a likely SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. The third component in the signaling pathway is D53, which shares predicted features with the class I Clp ATPase proteins and can form a complex with D14 and D3. SLs induce D53 degradation by the proteasome and abrogate its

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activity in promoting axillary bud outgrowth [174]. Analysis of mutants, such as D27, D17/CCD7, and D10/CCD8 from rice, showed that all these genes are involved in the biosynthesis of SLs. However, mutants lacking one or more of these genes are able to produce SL metabolites, which indicate an Int. J. Mol. Sci. 2016, 17, 1781 24 of 37 alternative minor pathway for SL biosynthesis [175]. The The first first identified identified function function of of SLs SLs in in higher higher plants plants was was as as aa stimulatory stimulatory signal signal in in seed seed germination such asas thethe witchweed Striga spp.spp. andand broomrapes (Orobanche and germinationof ofroot rootparasitic parasiticweeds, weeds, such witchweed Striga broomrapes (Orobanche Phelipanche spp.) [176]. The first germination stimulant identified for Striga was called strigol, a member and Phelipanche spp.) [176]. The first germination stimulant identified for Striga was called strigol, a of SLs isolated from cottonfrom (Gossypium L.) root exudates other[177]. compounds with member of SLs isolated cottonhirsutum (Gossypium hirsutum L.)[177]. root Many exudates Many other similar structures and with germination stimulatory properties were discovered in root exudates of compounds with similar structures and with germination stimulatory properties were discovered in host plant species [178]. Orobanche and Phelipanche spp. are obligate non-photosynthetic holoparasites root exudates of host plant species [178]. Orobanche and Phelipanche spp. are obligate nonthat depend fully on their host fordepend nutritional It is notfor clear how these parasites perceive SLs. photosynthetic holoparasites that fullyneeds. on their host nutritional needs. It is not clear how Recently, it has been suggested that MAX2, AtD14, or both, which are sensitive to SLs, might be these parasites perceive SLs. Recently, it has been suggested that MAX2, AtD14, or both, which are involved in this process [179]. In rice, the obstruction of the carotenoid biosynthesis in different sensitive to SLs, might be involved in this process [179]. In rice, the obstruction of the carotenoid steps of the pathway, including those to CCD7 substrates, led to reduced SLs production and biosynthesis in different steps of theleading pathway, including those leading to CCD7 substrates, led to secretion into the rhizosphere, resulting in decreased Striga germination and consequently lower Striga reduced SLs production and secretion into the rhizosphere, resulting in decreased Striga germination infection [180]. and consequently lower Striga infection [180].

Figure 4. Biosynthetic pathway of strigolactones (SLs) from β-carotene. Figure 4. Biosynthetic pathway of strigolactones (SLs) from β-carotene.

The analysis of genomes from several plant species showed a single copy for the CCD7 gene. The analysis of genomes from several plant species showed a single copy for the CCD7 gene. However, in most of the analyzed genomes, CCD8 is present as several copies [144]. In some cases, However, in most of the analyzed genomes, CCD8 is present as several copies [144]. In some cases, as in apple (M. domestica) and camelina (Camelina sativa), CCD8 genes are organized in tandem in the as in apple (M. domestica) and camelina (Camelina sativa), CCD8 genes are organized in tandem in same chromosome (Tables S2 and S3). CCD7 and CCD8, as other members of CCDs genes, contain the same chromosome (Tables S2 and S3). CCD7 and CCD8, as other members of CCDs genes, introns, with the exception of the NCED related group, which is intron-less. The CCD7 genes are contain introns, with the exception of the NCED related group, which is intron-less. The CCD7 characterized by the presence of 4–7 introns, four of which are well conserved among different genes are characterized by the presence of 4–7 introns, four of which are well conserved among plant species. The number of introns in CCD8 sequences ranges between 2 and 6, with 5 being the most frequent number among the species investigated. The identities among the CCD7 and CCD8 paralogous genes range between 36% and 100% and between 45% and 100%, respectively. The phylogenetic analysis of CCD7 proteins shows three clusters distinguishing the Embryophyte, Grass, and Eudicot phyla. However, a similar analysis with CCD8 proteins did not so clearly match this taxonomical classification (Figure S3).

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different plant species. The number of introns in CCD8 sequences ranges between 2 and 6, with 5 being the most frequent number among the species investigated. The identities among the CCD7 and CCD8 paralogous genes range between 36% and 100% and between 45% and 100%, respectively. The phylogenetic analysis of CCD7 proteins shows three clusters distinguishing the Embryophyte, Grass, and Eudicot phyla. However, a similar analysis with CCD8 proteins did not so clearly match this taxonomical classification (Figure S3). In contrast to the high functional conservation of the CCD7 and CCD8 enzymes, their genes present different expression patterns. Previous works in some plants, e.g., in rice, indicated that CCD7 and CCD8 genes are found to be upregulated in root [181]. In the columella cap of primary and lateral roots, CCD8 is highly expressed [ 182,183], and exogenous treatment with auxin 1-naphthaleneacetic acid (NAA) led to an overexpression of CCD8 expression in the primary root and cortical tissue. The expression of CCD8 in roots tissues from Arabidopsis [87], petunia [184], pea [185], kiwi [186], and tomato [187] exceeds shoot expression. However, in saffron and chrysanthemum, the opposite behavior was detected [188]; in rose, no transcripts are found in roots [189]. High expression of CCD7 in root from Arabidopsis is observed, [166], but more recently even higher transcripts were observed in seeds and in the stem vascular tissue [190]. Different patterns of CCD7 expression was found in rice [191], petunia [192], and tomato [170]. High expression was detected in vascular bundle tissue in rice, while CCD7 was mainly expressed in internodes and nodes in petunia, and CCD7 expression was low in green tissue in tomato in comparison to root and shoots. These differences suggest different mechanisms of the SL-regulation of the shoot branching of the root and shoot in different species. In addition to regulating shoot architecture and branching, SLs are involved in many other processes, including root growth, root hair elongation, lateral root formation, adventitious rooting, stem elongation, leaf expansion, leaf senescence, secondary growth, and drought and salinity responses [183,193–196]. CCD7 and CCD8 from soybean are involved in abiotic stress physiology, and their expression levels were greatly influenced by exogenous ABA treatment [112]. CCD7 from Lotus japonicus affects reproduction by reducing the number of flowers, fruits, and seeds and modulates leaf senescence and abscission [197]. CCD8 from kiwifruit modifies branch development and slows up leaf senescence [186]. An analogous association was detected in the rice tillering dwarf mutant D3, and the mutant MAX2/ORE9 from Arabidopsis [198]. Nevertheless, the delayed leaf senescence was not observed in the rms4 mutant from pea [199], suggesting that the connection between leaf senescence and branch development has followed a convergent evolution in plant species. Recent experimental evidence has revealed that auxin influences pea mycorrhizal symbiosis by regulating the production of SL plant hormones. During the symbiosis between the auxin deficient bsh mutant and the mycorrhizal fungus Glomus intraradices, the low auxin production correlated with reduced mycorrhizal colonization, SL levels, and biosynthetic gene expression, including CCD7 and CCD8 from pea [200]. In Crocus sativus, CCD7 and CCD8 were identified in saffron corms and stigmas, leading to attribute novel roles to SLs in this plant [201]. In corms, SLs act synergically with auxin to arrest the outgrowth of the axillary buds. In stigma tissue, transcripts were detected at a higher level than in vascular tissues, leaves, and roots. The abundance of both transcripts in immature orange stigmas suggests that these enzymes and SLs might regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm. A similar function was already suggested for SLs in flower development in petunia [184]. There has been great progress over the past years in characterizing plant CCD enzymes and their apocarotenoid products. As more functions are unraveled for apocarotenoids in plants, more additional roles will be assigned to CCD7 and CCD8. Further characterization of these genes may provide hints about the origin of parasitism, as well as new approaches for controlling parasitic plants and understanding important physiological processes of the underlying metabolic pathways.

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5. CCDs in Algae Algae are classified throughout many divisions of the kingdom Plantae. Their sizes range from single cells of picophytoplankton to seaweeds. Algae can synthesize many kinds of carotenoids that are absent in higher plants and therefore have been proposed as excellent chemotaxonomic markers [202,203]. Only few studies have been conducted with CCDs in algae. The best-known apocarotenoid in algae is retinal [204,205]. Most unicellular flagellate algae are phototactic [206]. They have developed an eyespot, which have been most studied in Chlamydomonas reinhardtii. The eye of Chlamydomonas comprises the optical system and at least five different rhodopsin photoreceptors. Two of these receptors are rhodopsin-ion channel hybrids switching between closed and open states by photoisomerization of an attached retinal chromophore [207]. The proteome of the eyespot apparatus in C. reinhardtii includes putative carotenoid cleavage dioxygenases homologous to Synechocystis sp. PCC 6803 ACOX_SYNY3 [208], which forms retinal from diverse apo-carotenoids in vitro [29]. However, the phylogenetic analysis of the CCD sequences present in the databases from algae did not show the presence of such homologues; instead, only CCD7 and CCD8 homologues have been identified (Table S2, Figure S4). As mentioned in the former section, SLs comprise an important class of apocarotenoid derivatives acting as hormones in plants [209] and have also been found in several algae [210]. A recent study showed that freshwater green algae belonging to the Charales, some of the closest freshwater green algal relatives of land plants, produce and exude SLs. The same study showed the presence of CCD7 and CCD8 homologues in different green algae taxa (Table S2, Figure S4) [211]. The authors suggested that SLs could play a role in rhizoid elongation in algae and thus increase their anchorage ability. Other important apocarotenoids in algae are the carotenoid-derived volatiles, which are released by diverse algae taxa and influence aquatic odors [212]. Ulva prolifera, Ulva linza, Monostroma nitidum, Ulothrix fimbriata, and Porphyra tenera produce volatile apocarotenoids in a high proportion. These C13 apocarotenoids or their derivatives exhibit growth-regulating properties in algae [213]. In addition, they may play ecological roles in providing competitive advantages, e.g., by inhibiting the growth of surrounding phytoplankton [214]. Functional carotenoid cleavage-like enzymes are expected to contribute to the formation of volatile apocarotenoids in these macro-algae [215]. No specific CCD has been isolated and identified as responsible for the production of these apocarotenoids, but the presence of homologous sequences closely related to CCD1 and CCD4 subfamilies (Table S2, Figure S4) suggests that theses CCDs could be good candidates to mediate the formation of these apocarotenoids volatiles. 6. Conclusions In nearly 20 years, the research on CCDs enzymes has evolved quickly, with the cloning of many different CCDs from plants and microorganisms. The increasing number of biochemical analyses for newly identified CCDs evidences the functional diversification of these enzymes in different species, adapted to a diversity of substrates and cleaving sites in a large variety of organisms. More recently, the knowledge on new putative CCDs is increasing enormously with the breakthrough of genome sequencing projects and transcriptome analyses of many additional species. The experimental basis for their future characterization has been solidly established, and the functional diversity found so far allows anticipating that new carotenoid or apocarotenoid substrates for novel CCD enzymatic reactions will be discovered in the next years. The biological purposes of such reactions will be predictably more elusive. Although the function of several novel CCDs has been resolved by in vitro analysis, we are far from understanding the activities and function of these enzymes in vivo in the different organisms, and more efforts will be needed to learn about how these CCDs are regulated at metabolic, transcriptional, and translational levels. The functions for newly identified CCDs and apocarotenoid products will be a future challenge, and effective research will require integrated multidisciplinary approaches to advance in this fascinating field of research. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/11/1781/s1.

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Acknowledgments: This work was supported by the Ibero-American network for the study of carotenoids as food ingredients (IBERCAROT, CYTED 112RT0445), the Spanish Government, Ministry of Science and Technology (projects AGL2015-70218, BIO2013-44239-R, AGL2012-34573, BIO2012-39716, and BIO2009-11131), the Andalusian Government (projects P07-CVI-02813 and CTS-6638), and the Generalitat Valenciana (PROMETEOII/2014/027). Author Contributions: All authors reviewed the literature and wrote this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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