Purine and Pyrimidine Phosphoribosyltransferases: A Versatile Tool ...

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REVIEW ARTICLE

Purine and Pyrimidine Phosphoribosyltransferases: A Versatile Tool for Enzymatic Synthesis of Nucleoside-5′-monophosphates J. Del Arco1 and J. Fernández-Lucas1,2,* 1

Applied Biotechnology Group, European University of Madrid, Urbanización El Bosque, Calle Tajo, s/n, 28670, Villaviciosa de Odón, Spain; 2Grupo de Investigación en Desarrollo Agroindustrial Sostenible, Universidad de la Costa, CUC, Calle 58 # 55 - 66. Barranquilla, Colombia Abstract: Background: In recent years, enzymatic methods have shown to be an efficient and sustainable alternative for the synthesis of nucleosides and nucleoside-5′-monophosphates (NMPs) to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. ARTICLE HISTORY Received: September 18, 2017 Accepted: October 13, 2017 DOI: 10.2174/1381612823666171017165707

Results: In this mini-review, we want to illustrate the application of phosphoribosyltransferases (PRTs) in enzymatic synthesis of NMPs. In this sense, many different examples about the use of PRTs as biocatalysts, as whole cells or enzymes, are described. In addition, it also includes detailed comments about structure and catalytic mechanism of described PRTs, as well as their possible biological role and therapeutic use, substrate specificity and advances in detection of new enzyme specificities towards different substrates. In addition, several examples about the use of PRTs in mono or multi-enzymatic synthesis of NMP analogues are shown. Finally, a brief discussion about advantages and drawbacks of the use of PRTs as industrial biocatalyst of NMPs has been commented. Conclusion: Despite the great potential of PRTs as biocatalysts for industrial synthesis of NMPs, several drawbacks must be overcome before reaching a suitable industrial application. In this sense, multi-enzyme systems provide an appropriate framework for this purpose. Moreover, future advances in different disciplines as protein engineering, bioinformatics and -omics will help to reach this goal.

Keywords: Biocatalysis, nucleic acid derivatives, phosphoribosyltransferase. 1. INTRODUCTION Nucleic acid derivatives, NADs (nucleosides, nucleotides and nucleobases) are essential metabolites in numerous biochemical processes. In this way, they are especially interesting for the treatment of several serious human illnesses, such 1as cancer, parasitic infections and viral diseases [1-3]. Most nucleoside analogues are prodrugs for nucleotides, generated by intracellular phosphorylation of the 5′-hydroxyl group to the corresponding mono-, di- or triphosphates. In this regard, the availability of synthetic nucleotide analogues allows the study of structure-activity relationships, kinetics and other metabolic consequences of nucleoside administration. As a consequence, the development of suitable and efficient methodologies for the synthesis of nucleoside-5'-mono- (NMPs) and diphosphates (NDPs) has been the target of several chemical and enzymatic synthetic efforts [4]. In addition, 2'-deoxynucleoside-5'monophosphates (dNMPs) are basic precursors of corresponding 2'deoxynucleoside-5'-triphosphates (dNTPs), which are widely used as chemical and biochemical reagents and in modern molecular biology experiments [5]. In this sense, the development of new synthetic processes of NMPs and dNMPs is the main focus area of pharmaceutical companies. Nucleotides are also used as additives in food industry. For example, some dietary nucleotides as Inosinic acid (Inosine-5'monophosphate, IMP) or Guanosinic acid (Guanosine-5'-monophosphate, GMP) are common additives used as flavour enhancer *Address correspondence to this author at the Applied Biotechnology Group, European University of Madrid, Urbanización El Bosque, Calle Tajo, s/n, 28670 Villaviciosa de Odón (Madrid), Spain; E-mail: [email protected] 1381-6128/17 $58.00+.00

in various foods, since they were found to induce an umami taste sensation [6]. In addition, the effect of dietary nucleotide supplementation on growth and immune function in term infants is extensively described [7]. As result of their importance, nowadays the demand for nucleotides in the food additives market is increasing, and the production of nucleotides has been well studied. Chemical synthesis of NMPs is usually performed by 5'phosphorylation of the precursor nucleoside. It often requires the use of chemical reagents (phosphoryl chloride, POCl3, or phosphorus pentoxide, P2O5), acidic conditions and organic solvents which are expensive and environmentally harmful [8, 9]. Indeed, chemical glycosylation of precursor nucleoside often requires timeconsuming multistep processes, including protection–deprotection reactions on the heterocyclic base and/or the ribose moiety, to allow the modification of natural occurring nucleosides [10]. The most habitual pathway to synthesize nucleoside analogues proceeds by coupling of the nucleobase to the ribo or 2'-deoxyribose moiety (or derivatives). In this sense, these methodologies need a glycosyl activation and protecting groups on the heterocyclic base and sugar moiety, as well as the exhaustive control of regio- and stereoselectivity of C-N glycosidic bond. Due to this, chemical synthesis of NMPs usually provides poor or moderate global yields, low product purity and are also associated with harsh reaction conditions and waste disposal issues [8, 11]. These drawbacks lead to a high price of the nucleosides and nucleotides, impeding their biological trials and studies, as well as limiting the wide therapeutic application. Due to the economical and social relevance of this kind of drugs, the availability of a stereoselective, sustainable and cheap synthetic methods is very relevant for the industry.

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The use of enzymes as industrial catalysts (white biotechnology) can thus provide novel and straightforward synthetic schemes and suitable and efficient methodologies for developing sustainable industrial processes. As a result, high quality products can be obtained by economically and technologically competitive processes. The use of enzymes in organic synthesis (biocatalysis) is thus increasing, particularly where industrial production has to deal with a high environmental impact (i.e. pharmaceutical), in an attempt to achieve a “sustainable chemistry”. In this frame, the enzymatic synthesis of nucleoside-5'-monophosphates (NMPs) is an attractive field since biotransformations show many advantages, such as, onepot reactions under mild conditions, high stereo- and regioselectivity, and an environmentally-friendly technology. Many different enzymes have also been used as valuable catalysts for mono or multi-enzymatic synthesis of NMPs [9, 12, 13], including nucleoside kinases (NKs) [11, 14, 15], acid phosphotransferases (AP/ PTases) [16, 17], 5'-phosphodiesterases [18] or nucleobase phosphoribosyltransferases (PRTs) [19, 20]. This review exhaustively covers literature reports on this topic with the final aim of presenting nucleobase PRTs as efficient biocatalysts for the synthesis of natural and non-natural NMPs. 2. PURINE AND TRANSFERASES

PYRIMIDINE

PHOSPHORIBOSYL-

2.1. General Concepts and Mechanism Purine and pyrimidine phosphoribosyltransferases (PRTs) belong to phosphoribosyltransferases family. They catalyze the reversible transfer of the 5-phosphoribosyl group from 5-phospho-αD-ribosyl-1-pyrophosphate (PRPP) to purine and pyrimidine nucleobases or derivatives in the presence of Mg2+. They are involved in the formation of all C-N glycosidic bonds in NMPs, both by salvage and de novo biosynthetic pathways, and are essential for the synthesis of nucleic acids (RNA and DNA) in organisms [21-24]. About kinetic of the reaction, several mechanisms have been proposed according to experimental data. On the one hand, the transfer of the 5-phosphoribosyl group from PRPP to the corresponding nucleobase seems to be catalyzed following a sequential ordered mechanism, in which PRPP binding is followed by nucleobase attack, and PPi product is released first followed by the corresponding NMP [25-29]. Some examples are UPRTs from Bacillus caldolyticus, E. coli or Mycobacterium tuberculosis [25-27], OPRT from yeast [28] or HGPRTs from Homo sapiens and Tritrichomonas foetus [29, 30] among others. On the other hand, some PRTs, such as OPRTs from Plasmodium falciparum and S. typhimurium seem to follow a random sequential kinetic mechanism [31, 32]. Finally, a ping-pong mechanism was also proposed for some PRTs, such as UPRT from Baker’s yeast [33]. However, most accepted hypothesis is that reaction proceeds with ordered sequential binding of substrates and ordered release of products. Several mechanisms have been proposed to explain the catalytic activity of PRTs. Reaction mechanism of 5-phosphoribosyl transfer from PRPP to nucleobase appears to be similar to double displacement reaction identified for some type of glycoside hydrolases [34]. In this type of enzymes, reaction proceeds through either stepwise reaction by formation of a discrete riboxocarbenium intermediate (SN1-like pathway) or by concerted mechanism through a ribooxocarbenium ion-like transition state (SN2-like pathway). It has been reported the analysis of the transition-state structures of OPRTs from Homo sapiens (HsOPRT), Plasmodium falciparum (PfOPRTS) and Salmonella typhimurium (StOPRT) by KIEs and it could be observed that the transition state is developed following ribooxocarbenium ion formation in these enzymes [35, 36]. Due to the similarity among PRTs, a SN1-type reaction that leads to the formation of an unstable oxocarbenium ion intermediate is the most accepted hypothesis (Scheme 1), but since there is no evidence of

Del Arco and Fernández-Lucas

kinetic isotope experiments performed up to date about other PRTs the possibility of a SN2-type reaction cannot be discarded. It is interesting to note that PRPP binds to the enzyme in association with a divalent metal ion, usually Mg2+. A first Mg2+ chelates with the pyrophosphoryl group (metal site 1), whereas a second Mg2+ (metal site 2) or a basic amino acid helps to stabilize the pyrophosphoryl group. Moreover, this second Mg2+ or basic amino acid contributes to a properly orientation of the base for catalysis. PRTs display a singular quaternary structure, since no monomeric PRT proteins are known and in most cases, the PRT core fold is part of a dimer interface in which a basic residue from each monomer contacts Mg•PRPP in the neighboring subunit. However, despite these cross-subunit interactions, most of PRTs do not show a cooperative mechanism in Mg•PRPP binding. In addition, only a few PRT proteins, such as UPRTs from E. coli [36], Sulfolobus shibatae [37] and Sulfolobus solfataricus [38] display allosteric phenomena. 2.2. Structural Classification According to the three-dimensional structures of a high number of PRTs, they can be classified in two different structural classes, I and II. All described class I PRTs display a conserved 13-residue ‘fingerprint’ region (PRPP binding-motif) in their amino acid sequence, which typically comprises four hydrophobic residues followed by two acidic residues (which interact with the vicinal hydroxyl groups of the ribose phosphate moiety), two hydrophobic residues, and four small residues [40-43] (Fig. 1).

Fig. (1). Multiple sequence alignment of different purine and pyrimidine phosphoribosyltransferases. Adenine phosphoribosyltransferases from Thermus thermophilus (TtAPRT1, TtAPRT2), E. coli (EcAPRT) and Leishmania donovani (LdAPRT). 6-oxopurine phosphoribosyltransferases from Thermus thermophilus (TtHGXPRT, TtXPRT), Leishmania donovani (LdHGPRT, LdXPRT), Toxoplasma gondii (TgHGPRT), Bacillus subtilis (BsXPRT), Enterococcus faecalis (EfXPRT) and Escherichia coli (EcXGPRT). Orotate phosphoribosyltransferases from Plasmodium falciparum (PfOPRT) and E. coli (EcOPRT). Uracil phosphoribosyltransferases from Sulfolobus solfataricus (SsUPRT), Escherichia coli (EcUPRT) and Bacillus caldolyticus (BcUPRT). Sequences were aligned by CLUSTALΩ. Conserved PRPP motif are encircled. Asterisk (*), indicates single, fully conserved residues; colon (:), conservation of strong groups; period (.), conservation of weak groups.

Members of class I, share a core region composed by four- or five-stranded parallel β-sheet surrounded by three α-helices and a hood domain offers residues that complete the active site [27, 40-

Whole Cell Biocatalysts for the Preparation of Nucleosides and their Derivatives

43]. Some of these residues are located in four flexible and highly mobile loops (loops I–IV) which are present in different conformations depending on the binding of substrates or products. (Fig. 2). The PRPP-binding site comprises three loops, whose sequences are highly conserved: a ‘PPi loop’ between β2 and α2; a ‘flexible loop’ between β3 and β4; and a ‘PRPP loop’ between β5 and α3. In this regard, PRPP binds to the C-terminal of the central β sheet motif, packed between the PPi and PRPP loops [27, 40-43] (Fig. 2). Moreover, above the core fold, PRTs display a peculiar poorly conserved structure known as the hood, which completes the catalytic active site and participates in binding of purine and pyrimidine nucleobases [27, 40-43] (Fig. 2).

Fig. (2). Overall structure of hypoxanthine-guanine phosphoribosyltransferase from Trypanosoma cruzi complexed with PRPP and hypoxanthine (sticks), and Mg2+ (black sphere) (PDB access code 1TC2). Black dotted lines encircle PRPP-binding site loops and hood domain.

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On the contrary, members of class II, do not share the conserved PRPP-binding motif in their amino acid sequences and the overall protein structure seems to be different to class I PRTs [44]. It is interesting to note that there is only one known member of this family, the quinolinic acid phosphoribosyltransferase. There are few examples about this enzyme, such as quinolinic acid phosphoribosyltransferase from Salmonella typhimurium or Mycobacterium tuberculosis among others. Regarding to purine and pyrimidine PRTs, all described purine or pyrimidine PRTs belong to type I PRTs. Representative members of this family are UPRT from Toxoplasma gondii [45], HGPRT from Toxoplasma gondii [27], HGXPRT from Pyrococcus horikoshii [46] or OPRT from Escherichia coli [47]. 2.3. Purine Phosphoribosyltransferases Purine PRTs catalyze the reversible transfer of the 5-phosphoribosyl group from PRPP to N9 in 6-amino or 6-oxopurines, such as adenine (1), hypoxanthine (2), guanine (3) and xanthine (4), in the presence of Mg2+ to synthesize adenosine-5'-monophosphate, AMP (5), inosine-5'-monophosphate, IMP (6), guanosine-5'monophosphate, GMP (7) or xanthosine-5'-mono-phosphate, XMP (8) respectively, in the presence of Mg2+ [27] (Scheme 2). The specificity of PRTs varies depending upon the nature of the specie, but in all reported cases, activity towards adenine is distinct from activity over hypoxanthine, guanine or xanthine. According their substrate specificity, purine PRTs are classified into two general classes: 6-aminopurine PRTs (APRT), strictly specific for 6aminopurines, such as adenine, 2-fluoroadenine (9) or 2chloroadenine (10) (Scheme 3) [27, 48, 49], and 6-oxopurine PRTs (HPRT, GPRT, XPRT, HGPRT or HGXPRT), that can recognize different 6-oxopurines, such as hypoxanthine, guanine, xanthine and other 6-oxo and 6-mercaptopurine analogs [19, 20, 27, 41]. 2.3.1. Biological Role and Therapeutic Use of Purine PRTs Purine metabolism is a metabolic route of vital importance in all living organisms, since purines are essential for the synthesis of nucleic acids (DNA and RNA), proteins and other metabolites. In mammalian cells, purine nucleotides can be synthesized by two different pathways, de novo and/or salvage pathways. In the de novo pathway cells use simple precursors like glycine, glutamine or aspartate for the synthesis of the different purine nucleotides. On

Scheme 1. Possible mechanism of PRTs. An SN1 mechanism passing through a discrete oxocarbenium intermediate (SN1-like pathway) could explain the scission of C-O bond of PRPP and the formation of the NMP. There are two transition states, one for the PPi departure to form oxacarbenium ion, and other one for the nucleophile approach performed by the nucleobase. An SN2 mechanism through a highly oxocarbenium ion-like transition state (SN2-like pathway) could also explain the hydrolysis of PRPP and formation of NMP. In this case, there is only one transition state, with the leaving group departure being concerted with, though in advance of, the nucleobase approach.



Scheme 2. Enzymatic synthesis of purine nucleoside-5'-monophosphates catalyzed by purine PRTs.

Scheme 3. Substrate specificity of purine PRTs over purine derivatives. the contrary, the salvage pathway is composed by a group of reutilization routes by which the cell can satisfy its purine requirements from endogenous and/or exogenous sources of preformed purines. In contrast to their mammalian hosts, most studied parasites lack the de novo pathways for purine biosynthesis and rely on the salvage pathways to satisfy their purine demands (Fig. 3). Since protozoan parasites lack de novo pathway, purine salvage enzymes are attractive targets for the design of inhibitors against protozoa, and the use of purine PRTs as therapeutic target it has been extensively reported [21, 50]. Due to this, most reported purine PRTs are purine PRTs from Trypanosoma brucei, T. cruzii, Leishmania species, Plasmodium falciparum or Toxoplasma gondii among others. Human and E. coli purine PRTs, are also extensively studied as reference model [19-21, 27]. As it is shown in Fig. 3, PRTs use the purine bases from hydrolysis of nucleosides by nucleoside phosphorylases (PNP or MTAP), nucleoside hydrolases (NH) or purine 2' deoxyribosylIt is described that human purine PRTs do not act over xanthine substrates, so XPRT has been exploited to develop antiparasitic drugs, such as allopurinol, aminopurinol or thiopurinol [21]. Moreover, selective inhibition of HGPRT from Plasmodium falciparum (PfHGPRT) by acyclic nucleoside phosphonates, immucillin 5'phosphates, iminoribitol derivatives and 2-(phosphoalkoxy) alkyl purine and pyrimidine derivatives leads to consider PfHGPRT as therapeutic target for Malaria treatment [50-52]. In addition, several

acyclic nucleoside phosphonate analogs were tested as inhibitors of HGPRT from Trypanosoma brucei (TbHGPRT). These compounds displayed promising low Ki values, suggesting they could be used as selective inhibitors for TbHGPRT [53]. Finally, in a novel study Ansari et al. [43] established a correlation between in silico and in vitro test analysis against Leishmania HGPRT and inhibitors by mean of QSAR studies [54]. Furthermore, the potential of the divergence in substrate specificity of some purine PRTs is not exclusive to treat parasitic diseases, and several purine PRTs have been used in suicide gene therapy for the cancer treatment. Trudeau et al. [55] performed the transfection of lung cancer cells with a suicide gene that encodes HGPRT from Trypanosoma brucei (TbHGPRT) to catalyze the conversion of allopurinol to a cytotoxic metabolite (Fig. 4). More recently, Parker et al. [56] has studied the effect of expression of excess levels of human APRT on the in vivo anti-tumor activity of prodrugs activated by E. coli PNP. 2.3.2. Adenine Phoshoribosyltransferase Adenine phosphoribosyltransferase, APRT (EC 2.4.2.7) catalyzes the transfer of the 5-phosphoribosyl group from PRPP to N9 in 6-aminopurines, such as adenine (1), or 6-aminopurine derivatives (Scheme 2). Adenine phosphoribosyltransferases are found in mammals but are less abundant than the 6-oxopurine enzymes. It is reported that APRT seems to be homodimeric, as it is described for several bacterial, archaea, fungal, protozoan and eu-



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Fig. (3). Purine salvage pathway in Trypanosoma brucei. NH, nucleoside hydrolase; PDT, purine 2'-deoxyribosiltransferase; AK, adenosine kinase; MTAP, S-methyl-5'- thioadenosine phosphorylase; Gua-DA, guanine deaminase; 5'-NT, nucleosidase; NK, nucleosido kinase; Ado-DA, adenosine deaminase; Ade-DA, adenina deaminase; Guo-DA, guanosina deaminase; Gua-DA, guanina deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; XPRT, xanthine phosphoribosyltransferase; AMP DA, adenosine-5'-monophosphate deaminase; S-AMP lyase, succinyladenylate lyase; S-AMP synth, succinyladenylate synthetase; GMP reductase, guanosine-5'-monophosphate reductase; IMP dehyd; inosine-5'-monophosphate dehydrogenase; GMP synth, guanosine-5'monophosphate synthetase.

karyotic APRTs, such as APRT from E. coli [57], Sulfolobus solfataricus [58], Saccharomyces cerevisiae [59], Leishmania donovani [60] or human APRT [61]. In this sense, it seems that the oligomeric state of APRTs is highly conserved among different living organisms. As we can observe in Fig. 2, APRT is a first class PRT (or PRT type I). Multiple sequence alignment of 6-amino and 6-oxopurine PRTs from different thermophilic and mesophilic bacteria and protozoan characterized so far, reveals that in all cases enzymes display the conserved 13-residue ‘fingerprint’ region, PRPP binding-motif of PRTs type I (Fig. 1). All described APRTs, display two sequence-adjacent aspartic residues, as corresponding acidic residues involved in interaction with the vicinal hydroxyl groups of the ribose phosphate moiety from PRPP. In addition, the structural alignment of different APRTs reveal that residues involved phosphate binding are very similar in both, side-chain and main-chain conformation, among them [57]. Unfortunately, APRT1 from Thermus thermophilus (TtAPRT1) seems to be the only exception to this general tendency. Fig. 1 showed us that only one of two acidic residues, which interacts with hydroxyl groups from PRPP, are present in its amino acid sequence. Rehse and Tahirov [62] described that TtAPRT1 displays several structural features consistent with its predicted role as an adenine phosphoribosyltransferase. However, they could observe some structural variations around the active site against other described APRTs. Moreover, the presence of a second APRT encoded in Thermus thermophilus genome, TtAPRT2 [63] (Fig. 1), which display a PRPP binding-motif more similar to other reported APRTs, suggests that TtAPRT1 could not play a very important role in purine scavenging in T. thermophilus. According to their tridimensional structures APRTs can be classified into long and short forms, distinguished from each other by N- and C-terminal extensions and by sequence variation. In this regard, APRT from Leishmania. donovani is the typical longAPRT, while APRTs from E. coli, Saccharomices cerevisiae and Homo sapiens are representative members of short-APRT class.

Adenine is the typical substrate for APRTs, but other modified 6-aminopurines, such as 2,6-diaminopurine (11), 6-amino-2hydroxypurine (12), 6-methylpurine (13), zeatin (14), isopentenyladenine (15), benzyladenine (16), and 8-azaadenine (17) (Scheme 3), among others, has been reported as substrates for APRT [60, 6466]. Even more interestingly, Boitz and Ullman showed that APRT from Leishmania donovani, LdAPRT, was able to recognize hypoxanthine as a substrate but far less efficiently than adenine [67]. Despite the great promiscuity of APRTs for the synthesis of NMPs, there are only few examples about their use as biocatalyst in NMP analogues from 6-aminopurine derivatives. Hansen et al. [58] explored the versatility as biocatalyst of APRT from thermoacidophile archaeon Sulfolobus solfataricus, SsAPRT, concluding that SsAPRT showed the highest activity at pH 7.5–8.5, but interestingly had also a distinct peak of activity at pH 4.5. 2.3.3. 6-oxopurine Phoshoribosyltransferases 6-oxopurine phosphoribosyltransferases, HPRT, HGPRT, XPRT, HGXPRT and GXPRT (EC 2.4.2.8 and 2.4.2.22), catalyze the transfer of the 5-phosphoribosyl group from PRPP to N9 in 6oxopurines (Scheme 2), such as hypoxanthine (2), guanine (3), and xanthine (5), and 6-oxopurine derivatives. 6-oxopurine phosphoribosyltransferases are most abundant phosphoribosyltransferases in organisms [27, 68]. The presence of dimeric and tetrameric 6-oxopurine PRTs in solution has been described [42, 58, 68]. On the one hand, HGXPRT from Sulfolobus solfataricus (SsHGXPRT) [58] and GPRT from Giardia lamblia (GlGPRT) [69] are dimers. On the other hand, HGXPRT from Thermus thermophilus (TtHGXPRT) [68], XPRT from Thermus thermophilus (TtXPRT) [63] HGPRT from Toxoplasma gondii (TgHGPRT) [27], XGPRT and HPRT from E. coli (EcXGPRT and EcHPRT) [70], are described as tetramers. Moreover, it has recently been described a homohexameric HGXPRT [46]. It seems that the oligomerization state of the 6-oxopurine PRTs might play a role in catalysis and stabilization of the active conformation as well as in the overall stability of the protein.


Contrary to APRTs, there is no consensus about the nature of two typical sequence-adjacent acidic residues involved in the recognition of PRPP. Some 6-oxopurine PRTs, such as TtHGXPRT, TgHGPRT, or xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani (LdXPRT and LdHGPRT) [27, 60, 67, 68, 71] display a glutamic residue followed by an aspartic residue, whereas TtXPRT, EcXGPRT or BsXPRT display two aspartic residues (Fig. 1) [63, 70, 72]. In the same way that other PRTs type I, 6-oxopurine PRT structures usually show the common α/β fold and different flexible loops (loops I–IV). All described 6-oxopurine phosphoribosyltransferases (HPRT, GPRT, XPRT, HGPRT and HGXPRT) share a similar overall fold encompassing the PRPP/PPi and purine nucleobase binding sites and share similar substrate binding contacts.


As described for reported 6-oxopurine PRTs, purine binding is stabilized by a network of hydrogen bonds between a Valine and Lysine residues, and N1, exocyclic O6 and N7 of the purine ring. This is the major difference between 6-oxopurine PRTs and APRTs, and it has a strong influence in the different substrate specificity. 6-aminopurines are not recognized by 6-oxopurine PRTs due to the absence of any suitable residues that can interact with exocylic –NH2 from C6. On the contrary, APRTs display residues that are hydrogen bonded to exocyclic –NH2, e.g. the backbone carbonyl oxygen of Ala147 (referred to APRT from Sulfolobus solfataricus [73]) or Ala24 (referred to APRT from G. lamblia [74]) is hydrogen bonded to the N6 amino group. In the same way, APRTs cannot form hydrogen bonds with 6-oxopurines, because they have a keto-group in the 6-position of the purine base. In an exciting studio, Kanagawa et al. [75] performed a structural comparison of different 6-oxopurine PRTs and suggested a

Fig. (4). Gene-directed enzyme-prodrug therapy, GDEPT (or suicide gene therapy), for cancer treatment by the expression of: A) hypoxanthine-guanine phosphoribosyltransferase, HGPRT, B) bifunctional cytosine deaminase-uracil phosphoribosyl transferase, CD-UPRT.

Scheme 4. Proposed active site structure of V157A, Y173H double mutant E. coli HPRT.


new classification of 6-oxopurine PRTs. According their structural elements, 6-oxopurine PRTs could be sorted in two groups, I and II (it must not be confused with canonical classification of PRTs, type I and II). 6-oxopurine PRTs type II display a N-terminal extension with additional secondary elements and a long loop connecting the second α-helix and β-strand compared with the group I enzymes. Substrate specificity of 6-oxopurine PRTs varies among different organisms, and is closely related to the nature of nucleobase [49]. Due to high variety of reported 6-oxopurine PRTs (HPRT, HGPRT, XPRT, HGXPRT and GXPRT), it can be observed that are very promiscuous enzymes, active over a broad range of 6oxopurine analogues, so each case should be studied separately. Schimandle et al. [76] isolated HGPRT from Plasmodium lophurae (PlHGPRT) and tested its substrate specificity over a broad range of different purine derivatives, such as hypoxanthine, 6mercaptopurine (18), 6-thioguanine (19), allopurinol (20) and 8azahypoxanthine (21), among others. PlHGPRT displayed good activity values over hypoxanthine, 6-mercaptopurine and 6thioguanine (Scheme 3). Azahypoxanthine, allopurinol, and 8azaguanine (22) also showed activity with the PlHGPRT but at a significantly reduced rate (Scheme 3). Keough et al. [77] studied the differences in the specificity between the purified P. falciparum HGXPRT and human hypoxanthine–guanine phosphoribosyltransferase enzymes, in order to exploit PfHGPRT as therapeutic target in rational drug design. Authors concluded that HsHGPRT has similar specific activities with guanine and hypoxanthine, but no activity was detected with xanthine. In addition allopurinol proved to be a very bad substrate for HsHGPRT (KMapp=135 µM, kcat/KMap=0.0006 µs-1M-1). Interestingly, PfHGPRT displays high activity values with guanine, hypoxanthine and allopurinol, but it also can act over xanthine. Scism et al. used a metabolically engineered E. coli strain that overexpressed a mutant variant of HPRT from E. coli (EcHPRT) for the preparation of a range of purine nucleotide analogues. Mutant EcHPRT, called 8B3PRT, was obtained as a product of directed evolution of hprt gene by error-prone PCR. Mutant variants were selected by an in vivo screening method growing transformants in minimal medium containing 1,2,4-‐triazole-3-carboxamide (TCA) (Scheme 3) (23) as substrate. Mutants harbouring efficient evolved HPRTs were able to use TCA to synthesize rivabirin-5'monophosphate. Once the optimal candidates were selected, sequencing of corresponding genes revealed a double mutation (V157A, Y173H), which leads to the enhancement of triazole phosphoribosyltransferase activity [19]. The first one, V157A, located in the active site nucleobase binding region, and the second one Y170H, distal to the active site (Scheme 4). In order to explore, the synthetic utility of 8B3PRT, authors examined the ability of this enzyme to accept different natural and non-natural purine bases. 8B3PRT demonstrated enhanced activity and high promiscuity in nucleobase recognition, processing a wide variety of nucleoside base analogues. Val157 is an essential residue in nucleobase binding, due to the hydrogen bonding of the N1 proton of hypoxanthine with backbone carbonyl group of Valine. It is possible that the V157A substitution modifies the structure of active site, and changes the nucleobase binding (Scheme 4). 8B3PRT showed significant enhanced activity (against wild type EcHPRT) with substrates with an electronegative atom in the C6 on the purine base, such as hypoxanthine, 6-mercaptopurine (18), 6-thioguanine (19), allopurinol (20), 6-chloroguanine (24), 6bromopurine (25), 6-chloropurine (26), 6-bromopurine (27) (Scheme 3). This method can potentially provide a great variety of nucleotide analogs. Due to mesophilic enzymes are not suitable biocatalysts for industry (e.g. low activity and stability under aggressive reaction conditions, such us extreme pH values, elevated temperatures or the presence of organic solvents), the use of thermozymes is an inter-


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esting alternative to circumvent most habitual problems for the scale of the processes (e.g. viscosity of the medium and concentration of substrates). In this sense, several thermophilic 6-oxopurine phosphoribosyltransferases have been reported in the literature, such as HGPRTs from Thermoanaerobacter tengcongensis (TteHGPRT) [78] and Sulfolobus solfataricus (SsHGPRT) [58], HGXPRTs from Thermus themophilus HB8 (TtHGXPRT) [68] and Pyrococcus horikoshi (PhHGXPRT) [46], and XPRT from Thermus themophilus HB8 (TtXPRT) [63]. Among them, it should be remarked the unusual high activity and stability under alkaline conditions (pH range 8-11) of TtHGXPRT. This tolerance to alkaline environments allowed TtHGXPRT to perform the enzymatic production of purine 5'-NMPs, such us 5'-IMP or 5'-GMP, at high concentrations of low water-soluble purine bases [68]. 2.4. Pyrimidine Phosphoribosyltransferases Similarly to purine PRTs, pyrimidine PRTs catalyze the reversible transfer of the 5-phosphoribosyl group from PRPP to the N1 of pyrimidine bases, such as orotate (28) or uracil (29), in the presence of Mg2+ to synthesize orotidine-5'-monophosphate, OMP (30) or uridine-5'-monophosphate, UMP (31) (Scheme 5). According their substrate specificity, pyrimidine PRTs are classified in two general classes: uracil PRT (UPRT), very specific for uracil and derivatives [26], and orotate PRT (OPRT) that can recognize orotate and other pyrimidine derivatives as substrate [5, 28].

Scheme 5. Enzymatic synthesis of pyrimidine monophosphates catalyzed by pyrimidine PRTs.

nucleoside-5'-

2.4.1. Biological Role and Therapeutic Use of Pyrimidine PRTs Despite the salvage of both purine and pyrimidine bases takes place in many organisms, purine base salvage is more habitual than the other one. In this sense, de novo pyrimidine synthesis exists in most organisms, even those auxotrophic for purines. Pyrimidine phosphoribosyltransferases are essential enzymes in both, de novo and salvage pathways (Fig. 5). On the one hand, OPRT catalyzes the formation of OMP, an essential step in the de novo biosynthesis of pyrimidines. OMP is subsequently converted to UMP (common precursor of all pyrimidine nucleotides) by OMP decarboxylase (OMPDC). In most of prokaryotes and yeasts, OPRT and OMPDC, are two distinct enzymes, encoded by two separated genes [79-80], but in majority of eukaryotes a bifunctional enzyme, named UMP synthase in mammalians, accomplish both metabolic steps (OMP synthesis and conversion to UMP) [41, 81-83]. In lower organisms (several protists, yeasts and bacteria) OPRT has been described as a monofunctional enzyme, such as OPRTs from E. coli (EcOPRt) [79] or Saccharomyces cerevisiae (ScOPRT) [80] or a bifunctional enzyme (similar to mammalian UMP synthases), as is seen in Trypanosoma cruzi and Leishmania mexicana [81-83]. Because of their similarity to mammalian UMP synthases, they are currently named UMP synthases. However, while mammalian UMP synthases, display OPRT at the N terminus and OMPDC at the C terminus, these lower UMP synthases display the inverse order (OMPDC at the N terminus and OPRT at the C terminus) On the other hand, UPRT is a key enzyme in the pyrimidine salvage pathway, a metabolic low-cost alternative to de novo pyrimidine synthesis for nucleotide synthesis (Fig. 5).


Fig. (5). General pyrimidine pathway. NH, nucleoside hydrolase; NDT, 2'-deoxyribosiltransferase; AK, adenosine kinase; MTAP, S-methyl-5'-thioadenosine phosphorylase; UP, uridine nucleoside phosphorylase; PyNP, pyrimidine nucleoside phosphorylase; CytDA, cytosine deaminase; Cyd-DA, cytidine deaminase; 5'-NT, 5'nucleotidase; UPRT, uracil phosphoribosyltransferase; OPRT; orotate phosphoribosyltransferase; ODC, orotidine 5'-phosphate decarboxylase

Because of these key roles in nucleotide metabolism, pyrimidine PRTs have been considered an interesting target for many different diseases, such as cancer [84, 85], malaria, leishmaniasis, sleeping sickness or Chagas disease among others [21-24,26]. Plasmodium falciparum, a protozoan parasite responsible for malaria, lacks the enzymes for pyrimidine salvage and cannot realize the salvage of pyrimidines from the host. Due to this fact, P. falciparum OPRT (PfOPRT) is thus an essential enzyme for parasite survival [86]. Zhan et al. [87] published an exhaustive study about the difference between transition states of PfOPRT and Human OPRT (HsOPRT). It also published in this study, the effect of p-nitrophenyl β-D-ribose-5′-phosphate as inhibitor of both OPRTs, which gave Kd values near 40 nM. This knowledge about OPRTs transition states provided new insights into the design of novel potential antimalarials. Zhang et al. [23], tested the effect of different p-nitrophenyl riboside 5'-phosphate analogues over PfOPRT and HsOPRT. Unfortunately, despite these compounds showed high affinity to PfOPRT, none of them could kill a P. falciparum culture. Another inhibitor of PfOPRT is pyrazofurin, and despite it exhibits non-significant IC50 values, it does not inhibit the HsOPRT [88]. In addition, uracil and 5-fluorouracil (32) (Scheme 6) are shown as weak inhibitors of P. falciparum OPRT, but they are able to reduce P. falciparum in vitro growth [89-90]. Furthermore, 5-fluoroorotate (33) (Scheme 6) and derivatives were tested as an alternative substrate for PfOPRT and displayed similar kinetic parameters to orotate [86]. 5′-5FOMP is transformed to 5′-fluoro-2′-deoxy-UMP metabolite, which leads to inactivation of P. falciparum thymidylate synthase [86].

Scheme 6. Enzymatic synthesis of pyrimidine nucleoside-5'-monophosphates catalyzed by pyrimidine PRTs.


In a similar way, Donini et al. reported the crystal structures of OPRT from Mycobacterium tuberculosis (MtOPRT). These structures could be a useful tool for the structure-based drug design of potent enzyme inhibitors for Mycobacterium tuberculosis [91]. Since OPRT, together with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD), is one of 5-fluorouracil (5FU) metabolic enzymes, its use as therapeutic target in different cancer types has been reported [92-95]. OPRT converts 5-FU to 5fluoro-2′-deoxyuridine-5′-monophosphate, an active anticancer metabolite. In this sense several studies have examined the relationship between OPRT activity in tumoral cells and their sensitivity to 5-FU [92-95]. Moreover, different studies about the expression levels of OPRT in cancer cells have been performed to know if high grade malignancy is associated with higher expression of 5-FU metabolic enzymes [95, 96]. Regarding to the therapeutic use of UPRT, it has been reported its essential role in different parasite survival. Despite human putative UPRT has been cloned and expressed, it did not show any detectable UPRT activity (probably due to the absence of two amino acids in the uracil binding domain) [97]. Because of this, parasite, yeast and bacterial UPRTs are interesting therapeutic targets for the development of new antimicrobial drugs [22, 26]. In order to explore 5-fluorocytosine resistance in several variants of Candida albicans, Alloush and Kerridge reported partial purification and substrate specificity of UPRT from Candida albicans (CaUPRT) [98]. CaUPRT is able to recognize 5-fluorouracil as an alternative substrate. Experimental results led authors to conclude that the resistance to 5-fluorocytosine in C. albicans seems to be linked to the decrease or lack of activity of CaUPRT. Iltzsch and Tankersley [99] performed a structure-activity study, in which 100 compounds were evaluated as ligands of uracil phosphoribosyltransferase from Toxoplasma gondii (TgUPRT) to explore its significance as a chemotherapeutic target. Based on this study, Schumacher et al. tried to explain the basis of pyrimidine discrimination and prodrug binding in TgUPRT using different crystal structures of TgUPRT complexed with uracil, 5-fluorouracil and UMP [43]. Furthermore, in a very recent study Villela et al. [100] proved that the uprt gene disruption does not affect M. tuberculosis growth. This report indicate us MtUPRT is not an adequate therapeutic target against M. tuberculosis. However, the therapeutic use of UPRTs is not exclusive as antibiotic agent. There are many different examples about the use of UPRTs in cancer treatment. An interesting example, is the combined application of cytosine deaminase (CD) and UPRT in suicide gene therapy through a bifunctional suicide gene that encodes for cytosine deaminase and uracil phosphoribosyltransferase for the treatment of ovarian carcinoma [84] and small cell lung cancer [85] (Fig. 4). Another interesting alternative is the separated transfection and co-expression of E. coli CD and E. coli UPRT genes for the treatment of prostate and human colon cancer with 5-fluorocytosine [101, 102]. 2.4.2. Orotate Phoshoribosyltransferase Orotate phosphoribosyltransferase, OPRT (EC 2.4.2.10) is an essential enzyme in pyrimidine biosynthesis, and is involved in the synthesis of orotidine 5'-monophosphate (OMP) from orotate and phosphoribosyl pyrophosphate (Scheme 5). Monofunctional OPRTs have been described as dimers (e.g. OPRTs from E. coli K12 [79], Saccharomyces cerevisiae [80], or Mycobacterium tuberculosis [103]). Furthermore, in Plasmodium falciparum, OPRT and OMPDC form a bifunctional heterotetrameric enzyme complex in solution, containing two homodimeric subunits (OPRT)2-(OMPDC)2 [104-105]. More recently, French et al. [106] described an OMPDC-OPRT complex (in reverse order to mammalian UMP synthases) in Leishmania donovani. Crystallization data revealed an unusual tetrameric structure that exhibits substrate-controlled oligomerization.


OPRT structures also display the common α/β fold and the different flexible loops involved in substrate binding and catalysis. Similar to other type I PRTs, PRPP binding motif is conserved in amino acid sequences of all reported OPRTs, such as MtOPRT, PfOPRT, ScOPRT or HsOPRT [103, 107]. In addition, all described OPRTs share conserved lysine residues involved in the binding of a highly charged Mg2+-PRPP complex and orotate, and catalysis. Interestingly, multiple sequence alignment of reported OPRT amino acid sequences, reveals the presence of the conserved hood domain, two substrate binding pockets (I and II) and the flexible catalytic loop that forms the active site and shields it from the solvent molecules [107]. Crystallographic data showed a homodimeric enzyme with two active sites (composed by amino acids of both subunits) located in the interface between monomers. The catalytic flexible loop closure upon substrate binding occurs between adjacent subunits, indicating that the catalytic lysine residue in the loop plays this role in the active site of the adjacent monomer [103, 107]. Because of its essential role in de novo biosynthesis, many different studies about the substrate specificity of OPRTs have been performed [41, 86, 87, 108-110]. Unfortunately, for a synthetic purpose, the main focus of these studies was the inhibition of OPRTs. However, some of them shed a light about the structural features of pyrimidine nucleobase analogues required for binding to OPRTs and provide the basis for the rational design of new inhibitors of this enzyme [108-110]. 5′-fluorouracil (32), 5′-fluoroorotate (33) and derivatives have been shown to be efficient substrates for OPRTs [49, 86, 90-96, 111] and could be employed in the enzymatic synthesis of many different interesting antimetabolites. Despite the fact that only a few studies about synthetic applications of OPRTs have been reported, several OPRTs have displayed interesting features which makes them interesting candidates for industry. The great activity values displayed by OPRTs from Thermus thermophilus (TtOPRT, 300 Units/mgenz) [112] or Corynebacterium ammoniagenes (CaOPRT, 423 Units/mgenz) [113], or the high thermal stability from TtOPRT, are representative examples about their potential as biocatalysts.


9

2.4.3. Uracil Phoshoribosyltransferase Uracil phosphoribosyltransferase, UPRT (EC 2.4.2.9) performs the synthesis of uridine-5'-monophosphate (UMP) from uracil and phosphoribosyl pyrophosphate, in the presence of Mg2+ (Scheme 5). Due to UMP is a common precursor of all pyrimidine nucleotides, UPRT is an essential enzyme in the salvage pathways for pyrimidine nucleotide synthesis. Different oligomeric states have been reported for the distinct bacterial, archaeon and protozoan UPRTs. On the one hand, UPRT from Gram-positive thermophile Bacillus caldolyticus (BcUPRT) is described as a dimer [25], whereas UPRTs from E. coli (EcUPRT) and thermoacidophilic archaeon Sulfolobus solfataricus (SsUPRT), are described as a trimer [114] and tetramer [115] respectively. On the other hand, UPRT from several protozoan parasites, such as Crithidia luciliae (ClUPRT) [116], Giardia lambia (GlUPRT) [117] and Toxoplasma gondii (TgUPRT) [43], have been described as dimers. However, TgUPRT has also been described as monomeric [118] or tetrameric (in presence of GTP, which affects the oligomerization state in solution) [45]. It seems that the oligomerization state of the PRTs could be essential in catalysis and stabilization of the active conformation as well as in thermal stability of the protein. [119, 46] Multiple sequence alignment of amino acid sequences of different UPRTs reveals a high similarity between different UPRTs and clearly suggests that their three-dimensional structures will be very similar, with a conserved core structure encompassing the PRPP/PPi and pyrimidine nucleobase binding sites and sharing similar substrate binding contacts (Fig. 6). In this regard, four typical short regions (I–IV) can be recognized, involved in substrate recognition, catalysis, and stability of the protein [26, 119, 120]. All described UPRTs contain typical conserved PRPP binding domain (region III). In addition, several conserved residues in regions I-II, involved in PRPP binding and stabilization, can also be identified in the different UPRTs. Near the end C-terminal, bacterial UPRTs display a highly conserved motif (region IV) which is critical in uracil-binding.

Fig. (6). Multiple sequence alignment of uracil phosphoribosyltransferases from Sulfolobus solfataricus (SsUPRT), Toxoplasma gondii (TgUPRT), Mycobacterium tuberculosis (MtUPRT), Escherichia coli (EcUPRT) and Bacillus caldolyticus (BcUPRT). Amino acid sequences were aligned by CLUSTALΩ. Amino acids for each polypeptide sequence were independently numbered. The secondary structural elements (dark grey cylinder for α helices and black arrows for β strands) for the SsUPRT, are indicated above the aligned amino acid sequences. Conserved regions (I-IV) are encircled. Asterisk (*) indicates single, fully conserved residues; colon (:) indicates conservation of strong groups; and period (.) indicates conservation of weak groups.



Scheme 7. Proposed active site structure of A) uracil phosphoribosyltransferase from Toxoplasma gondii (TgUPRT) complexed with 5-fluorouracil, B) TgUPRT in presence of 5-chloro, 5-bromo, or 5-iodouracil, and C) uracil phosphoribosyltransferase from Thermus thermophilus (TtUPRT) with thymine or different 5-halogenated uracil derivatives.

Traditionally, UPRTs show a remarkable preference for uracil as substrate, but neither thymine nor cytosine can be recognized by UPRTs. However, several bacterial, protozoan and plant UPRTs, such as uracil phosphoribosyltransferase from Eschericha coli, Leishmania donovani and Arabidopsis thaliana, have shown low activity over different pyrimidine derivatives, such as 5-fluorouracil (30), 4-thiouracil (34) or 6-azauracil (35) (Scheme 6) [49, 121]. Unfortunately, an extensive functional characterization of their potential as biocatalysts has not been carried out to date. Itzsch and Tankerslay [122] performed a structure-activity study, in which 100 compounds were evaluated as ligands of uracil phosphoribosyltransferase from Toxoplasma gondii (TgUPRT) by examining their ability to inhibit UMP synthesis. According with this work, larger volumes at position 5 of pyrimidine ring causes a steric hindrance in the active site, so large exocyclic substituents at this position decrease (fluoro) or abolish (methyl, chloro, bromo, iodo) base binding. Schumacher et al. [43] shed a light when they reported the structures TgUPRT both in Apo and bonded with uracil and 5-fluorouracil (PDBs 1bd3, 1bd4 and 1fpU respectively). The structural alignment of 5-fluorouracil complexed TgUPRT with uracil complex reveals minor adjustments in the active-site residues, especially in the base binding site (Scheme 7). As described for uracil, the molecule stacks between Met166 and Thr228 and forms four hydrogen bonds with active site residues, one water mediated. The main difference between the binding of uracil and 5fluorouracil is a small rotation in the latter in order to avoid steric clash with Ala168 Cβ (the distance between 5-fluoro and Ala168 Cβ is 3.1 Å). Consequently, the failure binding of 5-chloro (36), 5bromo (37) or 5-iodouracil (38) is related to the longer carbonhalogen bond and their larger van der Waals radius, which results in steric hindrance with Ala168 Cβ (Scheme 7). However, in a recent published work by Del Arco and co-workers, a different scenario is observed for Uracil phosphoribosyltransferase from Thermus thermophilus (TtUPRT) [123]. TtUPRT recognizes uracil and 5fluorouracil but is able to recognize 5-chloro-, 5-bromo-, 5iodouracil, thymine (39), and 5-hydroxymethyluracil (40) as substrates, due to an increase of the active-site size which avoid steric hindrance with larger volumes at position 5. 3. MULTI-ENZYMATIC SYNTHESIS OF 5-NMP ANALOGUES EMPLOYING PURINE OR PYRIMIDINE PRTS Enzymatic processes can involve one enzyme carrying out one specific reaction at a time, or multiple enzymes acting sequentially to yield a desired product. Multi-enzymatic cascade reactions offer several advantages, such as the realization of much more complex synthetic schemes, the ability to make reversible processes irreversible, the partial or total elimination of product inhibition prob-

lems or regeneration of cofactors for enzyme biocatalysis [12]. One of the most significant limitations to the practical application of PRTs as industrial biocatalysts is the high cost and instability of PRPP. To address these issues, several attempts to produce PRPP from non-expensive substrates have been reported. In this sense, it is very common the use of PRTs in multi-enzymatic cascade reactions, including different PRPP sources [20, 48]. Multi-enzymatic systems are can be used as in vitro or in vivo systems. On the one hand, Esipov et al. [48] designed a multi-enzymatic thermostable platform to synthesize modified NMPs from Dpentoses. In this regard, authors constructed an artificial biosynthetic pathway for the production of 2-fluoroadenosine-5-monophosphate (2F-AMP) and 2-chloroadenosine-5-monophosphate (2Cl-AMP) using recombinant thermophilic enzymes from Thermus sp. 2.9 and T. thermophilus HB8, including: i) the phosphorylation of D-pentose to ribose-5-phosphate by a ribokinase (RK), ii) the conversion of ribose-5-phosphate to 5-phospho-α-D-ribosyl-1pyrophosphate (PRPP) by a phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and iii) the transfer of the 5-phosphoribosyl group from PRPP to 2-fluoro (9) or 2-chloroadenine (10) by an APRT (Scheme 8) [48]. Scism and Bachmann [20] developed an interesting modification to this synthetic pathway including an in vitro ATP regeneration system. In this study, authors reported the use of cross-linked enzyme aggregate (CLEA) to synthesize purine nucleotides from D-ribose. In this strategy, authors used a mutant 6-oxopurine PRT (8B3PRT), and ATP is regenerated by including an adenylate kinase (AK) and pyruvate kinase (PK), which uses phosphoenol pyruvate (PEP) as a source of activated phosphate [20]. The combined action of five-component cascade system afforded high yields of different NMPs, such allopurinol, 6-thiopurine, 6-chloropurine and purine-5'-monosphosphates. Another variant of this strategy is the combination of a ribokinase (RK), a phosphoribosylpyrophosphate synthetase (PPS), a phosphoribosyl transferase (PRT), a nucleotide monophosphate kinase (NMPK), and creatine phosphate kinase (CPK) or pyruvate kinase (PKM). This system offers the possibility to synthesize many different purine and pyrimidine NMPs depending on selected PRT [124-126]. NMP is phosphorylated by NMPK to reach nucleotide diphosphate (NDP), which is phosphorylated by the action of creatine phosphate kinase (CPK) or pyruvate kinase (PKM). This method allowed the efficient enzymatic synthesis of several isotopically labeled nucleotides such as, 5-fluorocytidine-5′-triphosphate (5FCTP), 5-fluorouridine-5′-triphosphate (5F-UTP) or 2fluoroadenine-5′-triphosphate (2F-ATP) [123, 124] and the fluorescent nucleotide analog 8-azaguanosine-5′-triphosphate [125].



11

Scheme 8. Three-component cascade system for the preparation of nucleotide analogues. TspRK, Thermus sp ribokinase; TtPPS, Thermus thermophilus phosphoribosylpyrophosphate synthetase; TtAPRT Thermus thermophilus adenine phosphoribosyltansferase.

Scheme 9. Proposed role of UPRT in the in vivo multi-enzymatic synthesis of Nikkomycin X and Z by Streptomyces ansochromogenes.

On the other hand, the use of in vivo systems for multi enzymatic synthesis of NMPs offers several advantages such as, reactions take place from low-prize substrates, precursor compounds are synthesized by host cells, which can provide PRPP and recycle enzyme cofactors, and the isolation of the enzymes is not required. Valino et al [5] performed the synthesis of several pyrimidine NMP and NDPs by one-pot multistep microbial systems from uracil and orotic acid using C. ammoniagenes cells. More interestingly, nikkomycin X and Z (peptidyl nucleoside antibiotics) have been produced by Streptomyces ansochromogenes. One essential step in this process, is the synthesis of UMP or 5′-phosphoribosyl-4-formyl-4imidazoline-2-one from uracil and imidazolone (41) catalyzed by an UPRT (Scheme 9) [127, 128]. Liao et al. improved nikkomycin Z production in Streptomyces ansochromogenes by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding [127]. CONCLUDING REMARKS Purine and pyrimidine phosphoribosyltransferases are essential enzymes in nucleoside metabolism with a great potential in the pharmaceutical and alimentary industry. Despite there are numerous studies about PRTs, most of them are focused in the study of their potential as therapeutic targets for the treatment of viral, bacterial and carcinogenic diseases, and their application as industrial biocatalyst has not been well-studied. Nucleobase PRTs could be very efficient biocatalysts in the synthesis of natural and modified NMPs and even can replace or complement traditional chemical methods leading to an improvement of cost-effectiveness ratio. Unfortunately, the high cost and

instability of PRPP limits their industrial application. However, the use of PRTs in multi-enzymatic cascade systems can overcome this drawback, leading to interesting synthetic strategies. In addition, genetic engineering and bioinformatics, could provide the bases to develop new activities over non usual substrates. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS This work was supported by grant SAN151610 from the Santander Foundation. Grant 2016/UEM8, from Universidad Europea de Madrid, is also acknowledged. REFERENCES [1] [2] [3] [4]

De Clercq E. Recent highlights in the development of new antiviral drugs. Curr Opin Microbiol 2005; 8(5): 552-560 Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 2002; 3(7): 415424 Parker WB. Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of Cancer. Chem Rev 2009; 109(7): 28802893. Dellafiore MA, Montserrat JM, Iribarren AM. Modified Nucleoside Triphosphates for In-vitro Selection Techniques. Front Chem 2016; 4 (18): 1-13.

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