Characterization of quinonoid-dihydropteridine reductase (QDPR) from the lower eukaryote Leishmania major

Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H(4)biopterin pools. LmQDPR shows good homology to metazoan quinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8-9 copies containing LmQDPR plus two other genes. QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H(4)biopterin throughout this family of human parasites.     

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.     

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.     

Leishmania donovani pteridine reductase 1: biochemical properties and structure-modeling studies

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors.     

Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.     

Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Crystal structure of human dihydrofolate reductase complexed with folate

The crystal structure of recombinant human dihydrofolate reductase with folate bound in the active site has been determined and the structural model refined at 0.2-nm resolution. Preliminary studies of the binding of the inhibitors methotrexate and trimethoprim to the human apoenzyme have been performed at 0.35-nm resolution. The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p-aminobenzoyl-L-glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al. (1987) FEBS Lett. 218, 178-184]. The geometry of the polypeptide chain around the folate binding site in the human enzyme is not consistent with conclusions previously drawn with regard to the species selectivity of the inhibitor trimethoprim [Matthews et al. (1985) J. Biol. Chem. 260, 392-399].     

Structures of Leishmania major pteridine reductase complexes reveal the active site features important for ligand binding and to guide inhibitor design

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Kinetic characterization of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis: a paradigm shift for ts activity and channeling behavior

This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s(-) (1), C. hominis TS activity is 10-40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.     

Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent and which differ in the orientation of the pteridine ring of the substrate in the binding site. The Asp26----Asn mutant of L. casei dihydrofolate reductase has been prepared by oligonucleotide-directed mutagenesis and studied by one- and two-dimensional 1H-NMR spectroscopy. NMR studies of the mutant enzyme--folate--NADP+ complex show that this exists to greater than 90% in a single conformation over the pH* range 5-7.1. The single conformation observed corresponds to conformation I (the 'methotrexate-like' conformation) of the wild-type enzyme--folate--NADP+ complex. These observations demonstrate that Asp26 is the ionizable group controlling the pH-dependence of the conformational equilibrium seen in the wild-type enzyme.     

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.     

Exploring the inhibitory activity of Withaferin-A against Pteridine reductase-1 of L. donovani

Withaferin A is an abundant withanolide present in Withania somnifera leaves and to some extent in roots. It has been known for its profound anti-cancer properties, but its role in counteracting the Leishmania donovani infection has to be explored. Pteridine reductase 1 (PTR1) is involved in pteridine salvage and an important enzyme for the parasite growth, which could be targeted for the development of an efficient antileishmanial drug. We employed molecular docking studies to identify the binding mode of withaferin A with PTR1 in silico. We further cloned, expressed, and purified PTR1 of L. donovani and performed the enzyme kinetics using the Michaelis–Menten equation and enzyme inhibition studies with withaferin A by plotting the Lineweaver–Burk graph, which followed an uncompetitive mode of inhibition. We also showed the inhibition of the enzyme in the crude lysate of treated parasites. Thus, our study contributes towards understanding the mode of action of withaferin A against L. donovani parasite.     

The subcellular localization of the pteridines in strain R-26 of Rhodopseudomonas spheroides

Pteridines were detected in strain R-26 of Rhodopseudomonas spheroides and the pteridine concentrations in subcellular fractions were measured by a sensitive fluorimetric method. The whole cell fraction contained nearly equal concentrations of pteridine and reaction center bacteriochlorophyll. The soluble subcellular fraction contained the majority of the pteridine and the photochemically active chromatophore preparations contained very little. No pteridine was detected in purified reaction center complex preparations. This subcellular localization of pteridines was not consistent with a pteridine functioning as the primary electron acceptor in this photosynthetic system.     

Comparative topography of the catalytic sites of chromosomal dihydrofolate reductases and that of the enzyme encoded in R 67 plasmid from E. coli

The trimethoprim-resistant dihydrofolate reductase specified in E. coli by plasmid R 67, when compared, with other enzymes catalyzing the same reaction, to have a dissimilar primary, secondary, tertiary and quaternary structure. In regard to the tertiary structure, we show here that the pteridine binding site, in the plasmid-encoded enzyme, has a geometrical similarity with that of other chromosomal specified reductases.     

Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.     

Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H(4)B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (k(cat)/K(m)) for LmPTR1 are similar with dihydrobiopterin (H(2)B) and quinonoid dihydrobiopterin (qH(2)B) as substrates and about 20-fold lower than LmQDPR with qH(2)B. In contrast, TbPTR1 shows a 10-fold higher k(cat)/K(m) for H(2)B over qH(2)B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H(4)B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H(4)B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H(4)B as a medium supplement. These cells grew normally with H(4)B, which spontaneously oxidizes to qH(2)B, but were unable to survive in the absence of pterin or with either biopterin or H(2)B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.