Hypoxanthine-guanine Phosphoribosyltransferases Research Articles

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine–guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

A natural complex product Yaocha reduces uric acid level in a live zebrafish model

Introduction: This study was aimed to assess uric acid (UA)-lowering effect and its possible mechanisms of a natural complex product Yaocha in a live zebrafish model. Methods: The zebrafish high UA model was established by feeding 5 dpf zebrafish with both an uricase inhibitor potassium oxonate at 10 mM and an UA synthesis precursor xanthine sodium at 0.5 mM for 24 h. Yaocha was administered to the high UA zebrafish through soaking at 3 various concentrations, with allopurinol as a positive control. UA level, xanthine oxidase (XOD) activity, and mRNA expression of hypoxanthine guanine-phosphoribosyltransferases transferase (HPRT1) and organic anion transporter 1 (OAT1) were measured. Results: Yaocha effectively reduced UA level and inhibited xanthine oxidase (XO) activity in the high UA zebrafish. Yaocha could be a potential therapeutics for hyperuricemia through up-regulating HPRT1 and OAT1 gene expression and suppressing XO activity. Discussion: These results suggested that Yaocha hold a potential for high UA prevention and therapy, possibly through inhibiting UA production and promoting urate secretion and purine conversion.

Evaluating Iso-Mukaadial Acetate and Ursolic Acid Acetate as Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase Inhibitors.

To date, Plasmodium falciparum is one of the most lethal strains of the malaria parasite. P. falciparum lacks the required enzymes to create its own purines via the de novo pathway, thereby making Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPT) a crucial enzyme in the malaria life cycle. Recently, studies have described iso-mukaadial acetate and ursolic acid acetate as promising antimalarials. However, the mode of action is still unknown, thus, the current study sought to investigate the selective inhibitory and binding actions of iso-mukaadial acetate and ursolic acid acetate against recombinant PfHGXPT using in-silico and experimental approaches. Recombinant PfHGXPT protein was expressed using E. coli BL21 cells and homogeneously purified by affinity chromatography. Experimentally, iso-mukaadial acetate and ursolic acid acetate, respectively, demonstrated direct inhibitory activity towards PfHGXPT in a dose-dependent manner. The binding affinity of iso-mukaadial acetate and ursolic acid acetate on the PfHGXPT dissociation constant (KD), where it was found that 0.0833 µM and 2.8396 µM, respectively, are indicative of strong binding. The mode of action for the observed antimalarial activity was further established by a molecular docking study. The molecular docking and dynamics simulations show specific interactions and high affinity within the binding pocket of Plasmodium falciparum and human hypoxanthine-guanine phosphoribosyl transferases. The predicted in silico absorption, distribution, metabolism and excretion/toxicity (ADME/T) properties predicted that the iso-mukaadial acetate ligand may follow the criteria for orally active drugs. The theoretical calculation derived from ADME, molecular docking and dynamics provide in-depth information into the structural basis, specific bonding and non-bonding interactions governing the inhibition of malarial. Taken together, these findings provide a basis for the recommendation of iso-mukaadial acetate and ursolic acid acetate as high-affinity ligands and drug candidates against PfHGXPT.

In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.

Crystal Structures of Acyclic Nucleoside Phosphonates in Complex with Escherichia coli Hypoxanthine Phosphoribosyltransferase

AbstractDrug resistant uropathogenic E. coli associated with urinary tract infections (UTIs) are a serious and debilitating health threat. Therefore new drug targets to treat this disease need to be explored. One possible approach is to block the synthesis of the nucleoside monophosphates required for DNA/RNA production in these pathogens. In E. coli, the purine salvage pathway has two 6‐oxopurine phosphoribosyltransferases (PRTs), xanthine‐guanine PRT (EcXGPRT) and hypoxanthine PRT (EcHPRT). Here, we investigate acyclic nucleoside phosphonates (ANPs) as inhibitors of EcHPRT and have determined six crystal structures of EcHPRT in complex with ANPs. These data reveal the binding modes of these compounds and can assist in future rational structure‐based design efforts. It is suggested that a combination of inhibitors that block de novo and salvage pathways is a plausible approach to developing new antibiotics for E. coli UTIs. In addition, we provide details of a novel approach to accelerating the crystallization of this enzyme that may be of general applicability for rational drug discovery.

Thecis-peptide conformation at loop I of the Hypoxanthine phosphoribosyltransferases is not essential for the binding of substrates

The<i>cis</i>-peptide conformation at loop I of the Hypoxanthine phosphoribosyltransferases is not essential for the binding of substrates

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 Å resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Deltahgprt/Deltaxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084-16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Deltahgprt/Deltaxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2'-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Deltahgprt/Deltaxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Deltahgprt/Deltaxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Deltahgprt/Deltaxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Deltahgprt/Deltaxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Deltahgprt/Deltaxprt parasites displayed persistence and increased virulence in mice.

Crystal structure of a chimera of human and plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization

The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.

WITHDRAWN:Chimeric hypoxanthine guanine phosphoribosyltransferases: Effect of segment swapping on xanthine specificity and tetramer stability.

The paper entitled "Chimeric hypoxanthine guanine phosphoribosyltransferases: Effect of segment on xanthine specificity and tetramer stability", which was published online on 22 May 2006, was withdrawn at the author's request.

Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.

Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

Hypoxanthine phosphoribosyltransferases (HPRTs) are potential drug targets in the treatment of diseases caused by parasites. Also, defects in the human HPRT can result in gouty arthritis or Lesch–Nyhan syndrome. Active site loop I of HPRTs has been implicated in interactions between enzyme subunits that can influence the relative efficiencies of forward and reverse reactions, but the functional roles for invariant loop I residues (analogous with human Leu67 and Gly69) are poorly understood. Herein, saturation mutagenesis, complement selection, and steady-state kinetics were used to investigate the functional roles for Leu67 and Gly69. Seventy clones from a library of mutants were sequenced and more than 30 different mutations, or combinations of mutations, were identified. Several recombinant HPRTs with mutations at positions 67 and/or 69 supported the growth of a bacterial auxotroph on selective media, but only two of the mutants (L67M and G69S) could be recovered in the soluble fraction from bacteria induced to over-express the enzyme. The results of steady-state kinetic studies for L67M are consistent with the side chain of this residue participating in hydrophobic interactions between dimer subunits that are important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of PRPP, PPi, and hypoxanthine. The results for mutations at position 69 are consistent with only hydrogen or a small polar side chain being tolerated at this site. Kinetic studies of G69S suggest that side chains of residues at position 69 that project into the active site likely interfere with the binding of PRPP and PPi, as well as the positioning of a metal ion that indirectly influences the binding of purine bases and purine moieties of nucleotide substrates.

Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

Hypoxanthine phosphoribosyltransferases (HPRTs) are potential drug targets in the treatment of diseases caused by parasites. Also, defects in the human HPRT can result in gouty arthritis or Lesch–Nyhan syndrome. Active site loop I of HPRTs has been implicated in interactions between enzyme subunits that can influence the relative efficiencies of forward and reverse reactions, but the functional roles for invariant loop I residues (analogous with human Leu67 and Gly69) are poorly understood. Herein, saturation mutagenesis, complement selection, and steady-state kinetics were used to investigate the functional roles for Leu67 and Gly69. Seventy clones from a library of mutants were sequenced and more than 30 different mutations, or combinations of mutations, were identified. Several recombinant HPRTs with mutations at positions 67 and/or 69 supported the growth of a bacterial auxotroph on selective media, but only two of the mutants (L67M and G69S) could be recovered in the soluble fraction from bacteria induced to over-express the enzyme. The results of steady-state kinetic studies for L67M are consistent with the side chain of this residue participating in hydrophobic interactions between dimer subunits that are important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of PRPP, PPi, and hypoxanthine. The results for mutations at position 69 are consistent with only hydrogen or a small polar side chain being tolerated at this site. Kinetic studies of G69S suggest that side chains of residues at position 69 that project into the active site likely interfere with the binding of PRPP and PPi, as well as the positioning of a metal ion that indirectly influences the binding of purine bases and purine moieties of nucleotide substrates.

Interactions at the Dimer Interface Influence the Relative Efficiencies for Purine Nucleotide Synthesis and Pyrophosphorolysis in a Phosphoribosyltransferase

Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas' disease, show that the side-chain at position 68 can differentially influence the Km values for all four substrates as well as the kcat values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis–trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.

Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase

Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch–Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Δloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6–2.7-fold) changes in the Michaelis constants ( K ms) for substrates other than pyrophosphate (PP i), for which there was a 5.8-fold increase. In contrast, k cat values were severely affected by loop deletion, with rates that were 240–840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP i as a product of the salvage reaction.

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

Substrate Deformation in a Hypoxanthine-Guanine Phosphoribosyltransferase Ternary Complex: The Structural Basis for Catalysis

Background: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. Results: The crystal structure of T. gondii HGPRT with the substrate Mg 2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 Å resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre–transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. Conclusions: Three types of substrate deformation, chief among them an unexpected C2′- endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-π interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

Transition-state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases.

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.

Purine metabolism in Echinococcus multilocularis

The activities of the enzymes in Echinococcus multilocularis metacestodes involved in purine salvage were studied by HPLC. As in most parasites, this cestode relies entirely on salvage of preformed bases and nucleosides for its purine requirement. Therefore, these enzymes may be targets for drugs in the chemotherapeutic treatment of diseases caused by this parasite. The animals used in this study were gerbils ( Meriones unguiculatus). Enzyme activities from sera and hepatic tissue in control and infected animals were similar with the exception of adenine phosphoribosyltransferase which showed an activity 4-fold greater in the serum from control than in serum from infected animals. In the parasite, adenine and hypoxanthine-guanine phosphoribosyltransferases and adenosine deaminase had the highest activities. Therefore, in E. multilocularis metacestodes, this pathway seems to be important for the parasite’s metabolism.