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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. ??, pp. 1–xxx, ???? ??, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis*□ S

Received for publication, February 20, 2010, and in revised form, May 10, 2010 Published, JBC Papers in Press, May 27, 2010, DOI 10.1074/jbc.M110.115097

Giorgia Saga‡§, Alejandro Giorgetti§, Christian Fufezan¶, Giorgio M. Giacometti‡, Roberto Bassi§1, and Tomas Morosinotto‡2 From the ‡Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58 B, 35121 Padova, Italy, the §Dipartimento di Biotecnologie, Universita` di Verona, Strada le Grazie 15, 37134 Verona, Italy, and the ¶Institut fu¨r Biochemie und Biotechnologie der Pflanzen, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Hindenburgplatz 55, 48143 Mu¨nster, Germany

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Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.

In natural environments, light intensity is variable and often exceeds the saturation limit of photosynthesis (1, 2). As a consequence, excitation energy in excess may lead to production of reactive oxygen species and to oxidative stress, in a process called photoinhibition (2, 3). Photosynthetic organisms have evolved several mechanisms to dissipate excess energy safely and to increase the capacity for scavenging reactive oxygen species. A major role is played by the xanthophyll cycle (4, 5) in which the diepoxide xanthophyll violaxanthin is converted into the epoxide-free zeaxanthin. Zeaxanthin is a key molecule for plant photoprotection, being involved in singlet oxygen scavenging as well as singlet chlorophyll quenching (6 –10). Violaxanthin to zeaxanthin conversion is catalyzed by a lumenal enzyme, called violaxanthin de-epoxidase (VDE).3 The

* This work was supported in part by MIUR Grant PRIN 20073YHRLE and the University of Padova Grant CPDA089403 (to T. M.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1, Figs. 1 and 2, Equation, and an additional reference. 1 Supported by MIUR Grants PRIN 2008XB774B and FIRB RBIPO6CTBR. 2 To whom correspondence should be addressed. Tel.: 390498277484; Fax: 390498276300; E-mail: [email protected]. 3 The abbreviations used are: VDE, violaxanthin de-epoxidase; VDEcd, VDE central domain; WT, wild type; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid. □ S

ZSI

???? ??, 2010 • VOLUME 285 • NUMBER ??

reducing power for the reaction is provided by ascorbate (11), probably in its protonated form (12). VDE is activated when light-driven proton translocation across the thylakoid membrane exceeds the dissipation rate of the proton gradient by ATPase, leading to a decrease in pH in the thylakoid lumen. Inactive VDE is a soluble protein, but upon activation, it associates with the thylakoid membrane (13) where its substrate violaxanthin is located (14). When light intensity decreases, the stromal enzyme zeaxanthin epoxidase converts zeaxanthin back to violaxanthin (15, 16). Both VDE and zeaxanthin epoxidase have been suggested to belong to lipocalins, a multigenic protein family characterized by a conserved structural organization with an 8-strand ␤-barrel (15). VDE and zeaxanthin epoxidase are classified among outlier lipocalins because they do not present all three conserved regions typical of this multigenic family. Because of their rather low similarity with other lipocalins, their true membership of the lipocalin family has been challenged (17). In addition, VDE and zeaxanthin epoxidase are the only lipocalins, together with prostaglandin D synthase, that have enzyme activity, although most family members are involved in molecular transport (15, 18, 19). Beside the protein domain sharing similarity with lipocalins, which represents approximately half of the protein, VDE has two additional domains, with no clear homology to any other known protein, called the cysteine-rich and glutamate-rich domains (20, 21). The structure of the VDE putative lipocalin domain (VDEcd) was resolved by x-ray crystallography, showing that VDE is indeed a lipocalin with the typical conserved three-dimensional organization of an 8-strand ␤-barrel (22). That work also showed a pH-dependent conformational change associated with protein activation and dimeric organization at pH 5.0, which allows both violaxanthin rings to react at the same time (22). In this work, we investigated the VDE structure in more detail, with the aim of identifying the key residues involved in its catalytic activity. We first used in silico docking analysis to identify putative binding sites for its substrates violaxanthin and ascorbate. The importance of residues located in close proximity to the substrates was then accessed by mutation analysis. On these bases, we propose that two residues, Asp-177 and Tyr-198, are fundamental for the catalytic mechanism and we discuss similarities with other enzyme catalytic sites. JOURNAL OF BIOLOGICAL CHEMISTRY

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ARTNO: M110.115097

Identification of Violaxanthin De-epoxidase Active Site

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EXPERIMENTAL PROCEDURES Docking—The ligands, violaxanthin and ascorbic acid, were docked into the crystal structure of the active form of the VDE lipocalin domain (VDEcd) obtained at pH 5 (Protein Data Bank code 3CQR (22)), using AUTODOCK 4.0 (23). The parameters for the molecules were calculated by the AUTODOCK standard parameterization procedure. The Lamarckian Genetic Algorithm and 25 million energy evaluations per run were applied as a search method for the various docking results (for further details, see supplementary material). Before performing the docking procedure, we calculated the residue protonation state, i.e. the pKa values of the lipocalin domain (VDEcd), using MCCE version 2.4 (24) and DELPHI version 4 (25, 26). MCCE default parameters were used, and at least three independent runs were performed. The protonation state at pH 5 was thus calculated for all residues of VDE according to their calculated pKa value. An in-depth analysis of these results will be presented elsewhere.4 The following various combinations were considered for docking: (a) violaxanthin and dimeric VDE; (b) ascorbic acid and the VDE-violaxanthin complex; and (c) ascorbic acid and dimeric VDE. The potential grid map for each atom type was calculated by means of a cubic box centered in the putative binding cavity, with a distance of 0.375 Å between grid points. For each complex, 200 docking runs were performed, giving a total of 600 calculations. Ligand locations were then hypothesized by choosing the lowest energy conformations within the most densely populated clusters. In the case of the ascorbic acid docking experiments, the two most densely populated clusters were considered. Cluster analysis was based on the root mean square deviation distance among the ligands on each run (root mean square deviation cutoff, 2 Å). The minimal distances between violaxanthin, ascorbate, and the protein were calculated by means of p3d (27) and PyMOL (48). The latter was used to generate all images. Sequence Analysis—Protein VDE sequences from various plants (Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa) and diatoms (Phaeodactylum tricornutum, ID Phatr2 44635 Thalassiosira pseudonana, ID Thaps3 7677) were aligned with the Clustal algorithm. For the latter, the VDE sequences as identified in Ref. 34 were used. VDE Expression and Purification—The construct expressing mature A. thaliana VDE cloned in pQE60 was kindly provided by Prof. Yamamoto (21). For VDE expression, Escherichia coli cultures (Origami B strain (28)) with a 600-nm absorbance of 0.6 were induced with 1 mM isopropyl ␤-D-1-thiogalactopyranoside for 5 h at 37 °C. Cells were thereafter centrifuged at 6000 ⫻ g and 4 °C for 10 min, resuspended in Tris-HCl, pH 8, 250 mM NaCl, and lysed by sonication. VDE was then purified on a nickel affinity column (from Sigma). The fraction eluted with 100 mM imidazole was stored at ⫺80 °C. For CD spectra, larger protein amounts were obtained following the method in Ref. 14. Site-directed Mutagenesis—The VDE coding sequence was mutated with the QuikChange威 site-directed mutagenesis kit from Stratagene. 4

C. Fufezan, D. Simionato, and T. Morosinotto, manuscript in preparation.

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VDE Activity Test and HPLC—VDE activity was tested from the absorbance at 502 nm, where zeaxanthin and violaxanthin absorb light differently, and quantified as in Ref. 29. The reaction mixture contained about 1 ␮g of WT protein, 0.33 ␮M violaxanthin, 9 ␮M monogalactosyldiacylglycerol, and 67 mM citrate buffer, pH 5.1. The reaction was started with the addition of 60 mM ascorbate and after 20 min was stopped by adding 100 ␮l of 3 M Tris-HCl, pH 8.8. Carotenoids for HPLC analysis were extracted with diethyl ether; the organic phase was collected, dried in a SpeedVac, and resuspended in 80% acetone for HPLC analysis (30). Violaxanthin was purified from spinach by HPLC; monogalactosyldiacylglycerol was provided by Lipid Products, UK. SDS-PAGE and Western Blotting—Samples were run on 12% SDS-PAGE with a buffer system according to Ref. 31. Gels were stained with Coomassie Brilliant Blue R-250 or transferred to nitrocellulose membranes. VDE was detected by an antibody against His tag (from Sigma) or with homemade antibody raised against A. thaliana VDE (32). Membrane Binding Assays—VDE, overexpressed and purified as described above, was incubated with thylakoids as described previously (33). Briefly, 40 ␮l of partially purified VDE was added to 120 ␮g of chlorophyll of thylakoids and 40 ␮l of buffers with variable pH values (MES, HEPES, or Tris). Samples were incubated in the dark at 4 °C with mild agitation for 2 h and centrifuged at 13,000 ⫻ g for 10 min to precipitate thylakoids. The presence of VDE in supernatants (and pellets) was accessed by an antibody against the His tag, which thus specifically binds the recombinant protein.

RESULTS Docking of Violaxanthin in VDEcd Structure at pH 5—After VDEcd crystals at pH 5 had been obtained, one main objective was to reveal the structure of the enzyme-substrate complex. Unfortunately, all trials to obtain crystals of such a complex failed, because the substrate either inhibited crystal growth or did not bind to the protein previously crystallized. Analysis of protein packing within the crystals of the VDE lipocalin domain (VDEcd) showed that the barrel cavity, where the carotenoid violaxanthin is expected to bind, is rendered inaccessible by the interference of neighboring molecules, suggesting a reason for these failures. We therefore attempted the alternative approach of structural modeling to gain information on the violaxanthinbinding site and to characterize the structural determinants of the protein-active site. The violaxanthin molecule was docked in silico into the VDEcd structure at pH 5 by AutoDock 4.0. The docking calculations provided us with several clusters of conformations. The most densely populated (160 of 200 decoys), also corresponding to the conformations with lower energy values, allowed us to choose a preferred conformation. The latter was then funneled into a procedure of local optimization by the use of flexible side chains within the Autodock 4.0 software (see supplemental material). The lowest energy ligand-protein conformation is shown in Fig. 1; one violaxanthin molecule is bound to a VDE dimer, through a connection between the cavities of each monomer, a model consistent with a previous proposal (22). We also performed the same docking experiments VOLUME 285 • NUMBER ?? • ???? ??, 2010

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Identification of Violaxanthin De-epoxidase Active Site TABLE 1 Amino acid residues identified as closest to violaxanthin and their role in enzyme activity Minimal distances between amino acid residues and violaxanthin ring in the structural model of Fig. 1 are indicated. Right panel: all mutants were generated, and their enzyme activity is reported as determined with spectroscopic methods. Protein activity is expressed as % of WT control sample (ND means not detectable). Residue

Distance from violaxanthin

Mutation to

Activity (% WT)

Ala Ala

5⫾1 34 ⫾ 12

Ala Glu Leu Ala Ala Phe Ala Asn Ala Ala Gln Phe Phe

75 ⫾ 15 60 ⫾ 22 30 ⫾ 13 49 ⫾ 10 121 ⫾ 40 62 ⫾ 11 ND ND 56 ⫾ 23 ⬍2% ⬍2% ND ⬍2%

Å

C O L O R AQ: L

FIGURE 1. Model of violaxanthin docking in VDEcd at pH 5. A, VDEcd structure at pH 5 (Protein Data Bank 3CQR) is shown with docked violaxanthin molecule. Surface of amino acids is shown for protein, whereas violaxanthin is shown as orange spheres. One monomer is shown in gray and the other in light blue. B, same structure after 90° rotation; amino acid volume is 80% transparent, and chains are shown as blue ribbons. C, detail of structural model. Residues identified as close to violaxanthin ring (under 6 Å threshold) are shown as sticks and labeled according to numbering from A. thaliana mature protein.

using antheraxanthin, which also binds to the VDE dimer like violaxanthin (data not shown). Identification of Amino Acids Potentially Involved in Catalysis—To assess experimentally the validity of the in silico model, we tentatively identified residues potentially involved in ???? ??, 2010 • VOLUME 285 • NUMBER ??

His-121 Phe-123 Asn-134 Ile-135 Gln-153

4.0 4.3 5.7 3.4 2.4

Phe-155 Asn-167 Tyr-175 Asp-177

2.9 4.6 5.5 3.0

Asp-178 Trp-179

5.9 3.0

Tyr-198 Tyr-214

5.5 3.5

enzyme activity by selecting those located within a distance of 6 Å from the ring of the modeled violaxanthin molecule. Fig. 1C shows the residues identified; they are also listed in Table 1, together with their minimal distances from the violaxanthin ring. To complement structural information, we also analyzed VDE sequences from various alga and plant species. Among the former group, we considered in particular VDE protein sequences from diatoms, because they do not belong to Viridiplantae and are evolutionarily distant from vascular plants (34). They have a diadinoxanthin-diatoxanthin cycle but are also able to convert violaxanthin into zeaxanthin (35). Analysis of the diatom genome revealed that VDE from plants and diatoms shares a common evolutionary origin with one well conserved protein (34). In this case, we can assume that the key residues for catalysis are conserved. Fig. 2 shows a sequence alignment with the VDE lipocalin domain from various plant and diatom species; the protein sequences show a remarkable similarity, and 50 –55% of residues were completely conserved in plants and diatoms. From the alignment shown in Fig. 2 it can be inferred that conservation is even higher for residues identified as close to violaxanthin; 11 of 13 (85%) are identical in all species, which points to strong evolutionary pressure for their conservation and therefore an important role for enzyme activity or structural integrity. Mutational Analysis of Putative Residues Involved in Enzyme Activity—If the docking model is correct, we expect residues identified as the closest to the violaxanthin head group to play an important role in the de-epoxidation reaction. To verify this hypothesis, all 11 conserved residues were subjected to sitedirected mutagenesis. The rationale for amino acid substitution was to alter chemical properties (charge, polarity or aromatic nature) without major changes in their size. Table 1 lists all mutations performed. All mutant proteins were expressed in E. coli Origami cells and purified by affinity chromatography. The purified enzymes JOURNAL OF BIOLOGICAL CHEMISTRY

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Identification of Violaxanthin De-epoxidase Active Site ments with both the apo-VDE structure and the VDE-violaxanthin complex. Invariance of results indicates that ascorbate can access its binding site independently of the presence or absence of violaxanthin. We also verified that the inverse is true, i.e. violaxanthin docking is not significantly affected by the presence of ascorbate. Docking calculations with ascorbate yielded two densely populated clusters of 60/200 and 100/200 conformations, the former having slightly more favorable binding energies (Fig. 3 and supplemental Fig. 1). Because in silico data were not conclusive, we considered both clusters as putative ascorbate-bindFIGURE 2. Sequence alignment of VDE from various plant and diatom species. Clustal alignment of VDE ing sites (Fig. 3, A and B). It should sequences from various species, plants (At, A. thaliana; Nt, N. tabacum; and Os, O. sativa) and diatoms (Pt, P. be noted that in both cases ascortricornutum; Tp, T. pseudonana). Only region corresponding to lipocalin domain is shown. Numbers are from Arabidopsis mature protein. Residues identified to be within 6 Å of violaxanthin ring are boxed; stars indicate bate binds in a very similar position, amino acids putatively involved in ascorbate binding. although with different orientations. Analysis of interactions with were quantified by Western blotting to verify expression levels, the polypeptide chain showed that, in both clusters, ascorbate which were found to be similar for WT and all mutants. Because interacts with Thr-112, Asp-114, and Gln-119. Instead, intera fraction of expressed VDE is normally found insoluble after actions with Tyr-198 and Thr-245 are specific for only one of cell lysis, a reduction in purification yield was expected if the the binding conformations. All residues except Thr-112 are mutations caused strong alterations in protein folding. In our conserved in VDE sequences from various species, as shown in case, instead, the invariance of protein expression suggests that the alignment in Fig. 2, supporting their significance for protein activity. none of the mutations significantly affected protein folding. To validate the in silico calculations experimentally, all the The activities of mutant enzymes were measured, and the results are listed in Table 1. With the only exception of N167A, conserved residues were subjected to site-directed mutageneall mutations produced a significant effect on VDE activity. sis. The corresponding measured activities are listed in Table 2. This supports the idea that the identified residues have indeed Thr-112 was not mutated, not only due to its variability through been highly conserved through evolution, because of their the family of VDEs but also because its interaction with ascorimportance for enzyme activity. Nevertheless, not all mutations bate involved the protein backbone and was likely to be poorly altered enzyme activity equally; in the case of mutations on affected by any mutation. Strong reduction in activity was Asp-177 and Tyr-198, we did not detect any residual activity, observed for the residues that were proposed to interact with and although strongly reduced, mutants at sites Trp-179 and ascorbate in both models, Asp-114 and Gln-119, the mutation Tyr-214 still retained the ability to yield small amounts of zea- of the former leading to complete inactivation of the enzyme. xanthin. An additional group of mutants showed residual activ- However, Asp-114 has previously been suggested to be impority of around 35% or less with respect to WT, i.e. H121A, F123A, tant for VDE dimer stability (22), which prevented attributing and Q153L. All remaining mutants had activity in the range of loss activity only to ascorbate binding. Conversely, Gln-119 is 50 –75% of WT, with the already mentioned exception of located far from the violaxanthin epoxy group, is not engaged in N167A. In all cases, activity as verified by HPLC analysis was inter-monomer interactions, and is probably not involved in consistent with spectrophotometric assays. This was especially protein conformational changes, suggesting that the mutant important in the case of mutants with very low activity, because phenotype is indeed due to altered ascorbate binding. These data support the results on the ascorbate binding low amounts of zeaxanthin are better detected by HPLC. Ascorbate Docking into VDEcd Structure—The VDE catalytic region obtained in silico. The remaining mutants are useful for reaction requires ascorbate as the source of reducing power. distinguishing between the two possible binding conformaFor more insights on its binding site, we extended docking anal- tions. Y198F showed complete loss of activity, suggesting its yses to this second substrate. According to the literature, VDE great importance for ascorbate association; in the absence of should bind protonated ascorbic acid rather than ascorbate the tyrosyl OH group, ascorbate does not bind in the proper (12); however, docking experiments with both forms did not orientation and probably cannot provide reducing power to the reaction. Conversely, T245A showed activity equivalent to that yield significantly different results. Because there is no information on the order of binding of of WT, implying that this residue is not involved in the enzythe two substrates, we performed ascorbate docking experi- matic mechanism. These results thus support the hypothesis

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Identification of Violaxanthin De-epoxidase Active Site mutants analyzed above, we verified the ability of each mutant to associate with the membrane by incubatT112 T112 ing the protein at various pH values D114 D114 in the presence of a thylakoid memY198 brane preparation. At acidic pH values, VDE binds to the membrane T245 and can be precipitated by brief centrifugation, whereas unbound VDE Q119 Q119 remains in the supernatant (21, 33), where it can be detected by Western blotting (Fig. 4). In the case of WT, it is clear that VDE at pH 5 or below is C D hardly detectable in the supernatant, showing that it precipitates together with thylakoids. Fig. 4 shows the same test for the mutant proteins, revealing the greatest reduction in their activity; in all cases, membrane association occurs very similarly to that of WT, and at a pH below 5, the protein is bound to the membrane. These results clearly show that the strong decrease in activity was not due to impairment FIGURE 3. Ascorbate docking. A and B, two possible models were obtained from ascorbate docking in VDE of the membrane association. The structure at pH 5. In both cases, binding occurs in same part of the protein, but ascorbate orientation and interactions with polypeptide chain are different. Yellow, ascorbate; orange, violaxanthin. Residues interacting same test was performed for all with ascorbate are shown as sticks and labeled according to Arabidopsis mature protein. C, ascorbate-binding mutants analyzed in this work, and site confirmed by mutational analysis (model A). Protein, violaxanthin, and ascorbate carbon atoms are shown in no case did we observe alteration in gray, orange, and yellow, respectively. One monomer is shown in gray; the other is shown in light blue. D, zooming in binding cavity; structure is also rotated by 20°. Ascorbate and violaxanthin shown as sticks for of binding to the membrane (data clarity. not shown). These results confirm that the reduction in activity we TABLE 2 observed was indeed due to effects on the catalysis and/or bindActivity of mutants on residues putatively involved in ascorbate ing of substrates. In addition, these experiments provide supbinding port to the fact that all analyzed mutant proteins are correctly Mutants on residues identified as putatively involved in ascorbate binding in models shown in Fig. 3 are shown together their enzyme activity, indicated as % of WT (ND folded, i.e. if they had been misfolded, they would not have been means not detectable). able to bind to the membrane in a pH-dependent manner, a Residue Mutation Activity (% WT) capacity that requires a specific conformational change (37). Asp-114 Ala ND For further confirmation, we also measured the CD spectra of Asn ND Gln-119 Ala 32 ⫾ 10 inactive mutants, which showed that their folding was very simTyr-198 Phe ND ilar to that of WT (supplemental Fig. 2). Thr-245 Ala 82 ⫾ 35

A

C O L O R

B

that ascorbate binds as shown in the model in Fig. 3A. Fig. 3, C and D, shows the structural model of the VDE-ascorbate complex in more detail, which allows recognizing the presence of a small binding pocket where the ascorbate is located. It is worth noting that the identified ascorbate-binding site is located close to the violaxanthin ring, in a very good position to provide reducing power for the catalytic reaction. Assessment of Membrane Binding Capacity of Mutants— Violaxanthin is a hydrophobic molecule found dissolved in the thylakoid membrane. Thus, VDE, a soluble protein at neutral pH, needs to bind to the membrane to perform catalysis (16, 36). Therefore, it is possible that the observed reduction in mutant activity is a secondary effect, due to the inability of the enzyme to bind to the membrane, rather than an alteration in its catalytic activity. To exclude this possibility in any of the ???? ??, 2010 • VOLUME 285 • NUMBER ??

DISCUSSION Identification of VDE Active Site—In this work, we docked in silico violaxanthin and ascorbate to the VDE structure at pH 5 to build a model of the enzyme-substrate complex. This model was thereafter experimentally supported by site-directed mutagenesis, which confirmed positive identification of binding sites for both VDE substrates, i.e. violaxanthin and ascorbate. In silico and mutational analyses also allowed us to identify, among the residues lying close to the binding cavity, those directly involved in enzyme activity. Two residues were found to be essential for activity, Asp-177 and Tyr-198, and any change in charge/polarity caused complete loss of activity. In these mutants, no traces of antheraxanthin or zeaxanthin were detectable upon activity tests, even when prolonged well beyond standard reaction times (data not shown). It is worth recalling here that the effect of these mutations cannot be JOURNAL OF BIOLOGICAL CHEMISTRY

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esize that the reaction starts with protonation of epoxide oxygen, followed by a nucleophilic attack on the carbon. All previous considerations point to the identification of Asp-177 as responsible for the first reaction in VDE catalysis. It is worth noting that calculation of pKa values in the structure yielded a value of 5.8 for Asp-177, which is therefore mostly protonated at pH 5.4 Thus, Asp-177 is the best candidate to act as a proton donor to the violaxanthin epoxy group. This hypothesis also matches the violaxanthin binding model, which locates Asp-177 within 3 Å of the epoxy group (Fig. 5A). The other residue fundamental for activity is Tyr-198, which is located relatively far (7.6 Å) from the epoxy group. Other residues, like Trp-179, are found closer but are not as important for enzyme activity. However, the primary importance of Tyr198 for VDE activity is easily understood by analysis of the docking experiments; when ascorbate is bound in its pocket with the proper orientation, it forms a hydrogen bond with Tyr-198 and thus allows efficient electron donation to violaxanthin when the epoxy ring opens (Fig. 5A). Other Residues with an Important Role in VDE Activity—In addition to Asp-177 and Tyr-198, other residues play an important role in VDE activity, as their mutation greatly reduces enzyme activity, although small amounts of zeaxanthin are still produced. Among these residues is Trp-179, which contributes to violaxanthin binding by hydrophobic interaction, as explained by the fact that overpopulation of aromatic residues interacting with carotenoid molecules has been observed in many other carotenoid-binding proteins. A tryptophan molecule interacting with the head ring of a xanthophyll has been found in the LHCII structure (40), and interactions between carotenoids and aromatic residues have also been observed in photosynthetic reaction centers, bacterial light-harvesting proteins (41), and the orange carotenoid-binding protein (42). All these pigment protein complexes bind carotenoids, but they do not share any evolutionary relationship with each other, indiFIGURE 4. Membrane binding assay for VDE and mutants. Membrane cating that aromatic residues are particularly suitable for carotbinding capacity of mutants was tested by incubation at various pH values in enoid association. We speculate that the protein-carotenoid the presence of thylakoids membrane, as in Ref. 33. After incubation, thylasubstrate is a multicomponent interaction, and in the absence koids were precipitated together with any bound VDE, and the supernatant was analyzed by Western blotting with a His tag antibody. VDE was only of Trp-179, violaxanthin is still bound but probably with detected when binding to thylakoids was ineffective. Results shown cover WT reduced affinity and/or altered orientation, which explains the and mutants with the largest effect on enzyme activity. strong mutant phenotype. Tyr-214 is another aromatic residue with a major effect on VDE A B Electron activity. However, in this case, the Donation phenotype is not due to its aromatic Y198 nature but to the presence of an OH 32.1 group; its mutation into phenylalaAscorbate nine maintains its aromatic nature but still induces a drastic reduction 28.1 in protein activity. One explanation Violaxanthin for this phenotype is the fact that Tyr-214 is hydrogen-bound to HisProton D177 Donation 121 in the structure at pH 7, suggesting that it may be involved FIGURE 5. VDE active site. Details of model of Figs. 1 and 3 are shown, highlighting residues identified to be essential for VDE activity. A, Asp-177 and Tyr-198 are shown together with substrates ascorbate (above) and in the pH-induced conformational violaxanthin (below). Asp-177 is suggested to participate in the first step of reaction, involving nucleophilic change, like His-121 (22). attack on the epoxy ring. Tyr-198 is suggested to stabilize binding of ascorbate, which donates electrons to The next closest residues to vioepoxide. B, distances between Asp-177 and Tyr-198 in two monomers composing VDE dimer, respectively, 32.1 laxanthin in the structural model and 28.2 Å. attributed to disrupted protein folding, because protein expression and purification yields were substantially unaffected in the mutants with respect to WT, and membrane binding activity tests were unaltered (Fig. 4). It is highly unlikely that an unfolded protein can bind to thylakoid membranes in a pH-dependent manner, as binding requires a conformational change (37). Finally, inactive mutants also have CD spectra indistinguishable from WT (supplemental Fig. 2). It has previously been shown that pepstatin A, an inhibitor of Asp proteases, also affects VDE activity. This observation suggested that the reaction centers of VDE and aspartic protease have a common structural organization (37) and supported the first idea that an active aspartate is involved in the VDE catalytic mechanism (38). More importantly, other enzymes active in the degradation of epoxy groups, such as epoxide hydrolases, also rely on aspartate residues for epoxy cycle opening (39). In these hydrolases, the first reaction step involves a nucleophilic attack on the epoxy group which, in an acidic environment, is known to be sensitive to such an attack. In the case of VDE, we may hypoth-

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Identification of Violaxanthin De-epoxidase Active Site are Phe-123 (4.3 Å), Gln-153 (2.4 Å), and Phe-155 (2.9 Å). They are probably involved in enzyme-substrate complex stabilization, as their mutation induces only partial inactivation, and significant amounts of zeaxanthin are still produced. The case of Gln-153 is interesting because, in the structural model, the residue is involved in a putative hydrogen bond with violaxanthin, which may help to bind the carotenoid in its correct orientation. Consistent with the hypothesis that polar interactions with the substrate are significant, the strongest effect is observed when Gln is substituted with Leu. The consequences of mutations on these residues may be stronger in vivo, because in vitro activity tests are performed in excess of the substrates, i.e. in conditions that mask alterations in substrate binding affinity. A Dimeric Model for VDE—Structural data on VDE suggest that, in its active conformation, the protein is a dimer. This organization is unlikely to be a crystallization artifact, because the monomer-monomer interfaces are larger for VDE than for other lipocalins well known to be dimers (22, 43, 44). Our docking experiments support the identification of the violaxanthinbinding site in a VDE dimer, and mutational analysis is consistent with in silico calculations. In this respect, it is interesting to compare the structural model of violaxanthin binding presented here with experimental data on VDE activity in various carotenoid species. VDE has been shown to be active with carotenoids in all-trans configurations. Violeoxanthin, which is identical to violaxanthin except for the presence of a double bond in cis configuration, is not de-epoxidized by VDE, even on a single end of the molecule (29). Because the rest of the molecule is identical, this difference can only be due to the molecular shape of the carotenoid, which cannot fit into the VDE cavity. This result led to the estimation of 30 Å for the length of the violaxanthin-binding site (29). This is consistent with the dimeric model which, in the active form, contains a cavity having the size of a violaxanthin molecule, and the distances between the active sites are around 30 Å (Fig. 5B). Instead, in the case of a monomeric VDE, the cavity would be around 15 Å long, and violeoxanthin would be bound as well as violaxanthin. Several inter-chain interactions further support the dimeric model, and particular significance must be attributed to that between Asp-114 and Arg-138 in the companion monomer. Mutations in either of these two residues, impairing salt bridge formation, have been shown to abolish VDE activity completely (22). Our results clearly show that these two residues are neither in close proximity to the substrate nor to any other residue essential for the activity of VDE, and thus the involvement of this salt bridge in dimer stabilization is the most probable hypothesis explaining the strong mutant phenotype. All these considerations support the idea that the active VDE form is a dimer. However, we might still ask why this oligomeric state had never been observed before. The most probable answer is that VDE requires lipids for its activity (29, 45), whereas dimerization and lipid association are closely connected. This is consistent with the observation that we never succeeded in maintaining VDE in solution at low pH without lipids. However, unfortunately, the presence of large lipid par???? ??, 2010 • VOLUME 285 • NUMBER ??

ticles impairs determination of native molecular weight and thus of oligomeric state. Finally, the dimeric model raises the question as to how antheraxanthin forms. One explanation is that we identified two ascorbate-binding sites for one dimer. If one of these sites is empty then, upon violaxanthin binding, we would expect antheraxanthin to form instead of zeaxanthin. This fits two further considerations. First, it has been shown in isolated chloroplasts that ascorbate can be limiting for zeaxanthin formation, and in this case, we expect antheraxanthin to form (46). Second, the Km value for ascorbate is much larger than that for violaxanthin (1 mM versus 5 ␮M (12, 47)), so that one ascorbate-binding site might remain empty in a fraction of active sites. An alternative/additional explanation why the dimeric VDE fails to produce zeaxanthin alone is found in the above-mentioned in silico calculation, which indicates that Asp-177 has a pKa of 5.8 in the dimeric form.4 If protonation of this amino acid is essential for protein activity, we would expect that about 10% of the active centers not to be able to perform the reaction, with the consequent formation of antheraxanthin instead of zeaxanthin. However, antheraxanthin, which eventually forms, can still rebind to VDE and be converted into zeaxanthin. Acknowledgment—We thank Stefano Cazzaniga (Universita` di Verona) for help with HPLC analysis.

REFERENCES 1. Ku¨lheim, C., Agren, J., and Jansson, S. (2002) Science 297, 91–93 2. Li, Z., Wakao, S., Fischer, B. B., and Niyogi, K. K. (2009) Annu. Rev. Plant Biol. 60, 239 –260 3. Barber, J., and Andersson, B. (1992) Trends Biochem. Sci. 17, 61– 66 4. Demmig, B., Winter, K., Kru¨ger, A., and Czygan, F. C. (1987) Plant Physiol. 84, 218 –224 5. Gilmore, A. M., Hazlett, T. L., and Govindjee (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2273–2277 6. Niyogi, K. K., Grossman, A. R., and Bjo¨rkman, O. (1998) Plant Cell 10, 1121–1134 7. Holt, N. E., Zigmantas, D., Valkunas, L., Li, X. P., Niyogi, K. K., and Fleming, G. R. (2005) Science 307, 433– 436 8. Dall’Osto, L., Caffarri, S., and Bassi, R. (2005) Plant Cell 17, 1217–1232 9. Havaux, M., Dall’osto, L., and Bassi, R. (2007) Plant Physiol. 145, 1506 –1520 AQ: C 10. Dall’Osto, L., Cazzaniga, S., Havaux, M., and Bassi, R. (2010) Mol. Plant, 11. Yamamoto, H. Y., Wang, Y., and Kamite, L. (1971) Biochem. Biophys. Res. Commun. 42, 37– 42 12. Bratt, C. E., Arvidsson, P. O., Carlsson, M., and Akerlund, H. E. (1995) Photosynth. Res. 45, 169 –175 AQ: D 13. Hager, A., and Holocher, K. (1994) Planta 192, 581–589 AQ: E 14. Morosinotto, T., Baronio, R., and Bassi, R. (2002) J. Biol. Chem. 277, 36913–36920 15. Bugos, R. C., Hieber, A. D., and Yamamoto, H. Y. (1998) J. Biol. Chem. 273, 15321–15324 16. Jahns, P., Latowski, D., and Strzalka, K. (2009) Biochim. Biophys. Acta 1787, 3–14 17. Ganfornina, M. D., Gutie´rrez, G., Bastiani, M., and Sa´nchez, D. (2000) Mol. Biol. Evol. 17, 114 –126 18. Urade, Y., and Hayaishi, O. (2000) Biochim. Biophys. Acta 1482, 259 –271 19. Hieber, A. D., Bugos, R. C., and Yamamoto, H. Y. (2000) Biochim. Biophys. Acta 1482, 84 –91 20. Bugos, R. C., and Yamamoto, H. Y. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 6320 – 6325 21. Hieber, A. D., Bugos, R. C., Verhoeven, A. S., and Yamamoto, H. Y. (2002)

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AQ: G

AQ: H

Planta 214, 476 – 483 22. Arnoux, P., Morosinotto, T., Saga, G., Bassi, R., and Pignol, D. (2009) Plant Cell 21, 2036 –2044 23. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., and Olson, A. J. (1998) J. Comput. Chem. 19, 1639 –1662 24. Song, Y., Mao, J., and Gunner, M. R. (2009) J. Comput. Chem. 30, 2231–2247 25. Rocchia, W., Sridharan, S., Nicholls, A., Alexov, E., Chiabrera, A., and Honig, B. (2002) J. Comput. Chem. 23, 128 –137 26. Rocchia, W., Alexov, E., and Honig, B. (2001) J. Phys. Chem. B 105, 6507– 6514 27. Fufezan, C., and Specht, M. (2009) BMC Bioinformatics 10, 258 28. Prinz, W. A., Aslund, F., Holmgren, A., and Beckwith, J. (1997) J. Biol. Chem. 272, 15661–15667 29. Yamamoto, H. Y., and Higashi, R. M. (1978) Arch. Biochem. Biophys. 190, 514 –522 30. Gilmore, A. M., and Yamamoto, H. Y. (1991) Plant Physiol. 96, 635– 643 31. Laemmli, U. K. (1970) Nature 227, 680 – 685 32. Ballottari, M., Dall’Osto, L., Morosinotto, T., and Bassi, R. (2007) J. Biol. Chem. 282, 8947– 8958 33. Gisselsson, A., Szilagyi, A., and Akerlund, H. E. (2004) Physiol. Plant. 122, 337–343 34. Coesel, S., Oborník, M., Varela, J., Falciatore, A., and Bowler, C. (2008) PLoS ONE 3, e2896 35. Lohr, M., and Wilhelm, C. (1999) Proc. Natl. Acad. Sci. U.S.A. 96,

8 JOURNAL OF BIOLOGICAL CHEMISTRY

8784 – 8789 36. Rockholm, D. C., and Yamamoto, H. Y. (1996) Plant Physiol. 110, 697–703 37. Kuwabara, T., Hasegawa, M., Kawano, M., and Takaichi, S. (1999) Plant Cell Physiol. 40, 1119 –1126 38. Kawano, M., and Kuwabara, T. (2000) FEBS Lett. 481, 101–104 39. Arand, M., Cronin, A., Oesch, F., Mowbray, S. L., and Jones, T. A. (2003) Drug Metab. Rev. 35, 365–383 40. Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X., and Chang, W. (2004) Nature 428, 287–292 41. García-Martín, A., Pazur, A., Wilhelm, B., Silber, M., Robert, B., and Braun, P. (2008) J. Mol. Biol. 382, 154 –166 42. Kerfeld, C. A., Sawaya, M. R., Brahmandam, V., Cascio, D., Ho, K. K., Trevithick-Sutton, C. C., Krogmann, D. W., and Yeates, T. O. (2003) Structure 11, 55– 65 43. Campanacci, V., Bishop, R. E., Blangy, S., Tegoni, M., and Cambillau, C. (2006) FEBS Lett. 580, 4877– 4883 44. Lascombe, M. B., Gre´goire, C., Poncet, P., Tavares, G. A., RosinskiChupin, I., Rabillon, J., Goubran-Botros, H., Mazie´, J. C., David, B., and Alzari, P. M. (2000) J. Biol. Chem. 275, 21572–21577 45. Latowski, D., Akerlund, H. E., and Strzałka, K. (2004) Biochemistry 43, 4417– 4420 AQ: I 46. Neubauer, C., and Yamamoto, H. Y. (1994) Photosynth. Res. 39, 137–147 47. Havir, E. A., Tausta, S. L., and Peterson, R. B. (1997) Plant Sci. 123, 57– 66 AQ: J 48. DeLano, W. L. (2008) The PyMOL Molecular Graphics System, DeLano Scientific LLC, Palo Alto, CA

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