Phosphoribosyl-1'-pyrophosphate Research Articles

Crystal structures of anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae.

Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the transfer of the phosphoribosyl group of 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to anthranilate to form phosphoribosyl-anthranilate. Crystal structures of AnPRTs from bacteria and archaea have previously been determined; however, the structure of Saccharomyces cerevisiae AnPRT (ScAnPRT) still remains unsolved. Here, crystal structures of ScAnPRT in the apo form as well as in complex with its substrate PRPP and the substrate analogue 4-fluoroanthranilate (4FA) are presented. These structures demonstrate that ScAnPRT exhibits the conserved structural fold of type III phosphoribosyltransferase enzymes and shares the similar mode of substrate binding found across the AnPRT protein family. In addition, crystal structures of ScAnPRT mutants (ScAnPRTSer121Ala and ScAnPRTGly141Asn) were also determined. These structures suggested that the conserved residue Ser121 is critical for binding PRPP, while Gly141 is dispensable for binding 4FA. In summary, these structures improved the preliminary understanding of the substrate-binding mode of ScAnPRT and laid foundations for future research.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.

Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site.

Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile β2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The β2-α6 loop moves sequentially between a "folded" conformation, partially occluding the anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the β2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the β2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme.

Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis

AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP.

The Substrate Capture Mechanism of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Provides a Mode for Inhibition

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.

Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase

Nicotinamide (NM) phosphoribosyltransferase (NMPRTase) catalyzes the reaction of NM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form NM mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the (1)H and (31)P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0-2.2 A were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and the leaving group for the requisite inversion of configuration at the C1' carbon of the ribose ring. In the active site near the C1' atom of the bound PRPP or NMN, there was neither negatively charged group nor waterproof environment necessary to support the feasibility of a ribo-oxocarbocation intermediate inherent in the S(N)1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.

Presence, preliminary properties and partial purification of 5-phosphoribosylpyrophosphate amidotransferase from Artemia sp.

5′-Phosphoribosylpyrophosphate amidotransferase, which catalyzes the synthesis of phosphoribosylamine in the de novo synthesis of purine nucleotides, has been detected and partially purified approx . 800-fold from Artemia sp. nauplii. The apparent K m values for 5′-phosphoribosyl 1-pyrophosphate as substrate were 0.7 mM and 0.4 mM in the presence of glutamine and ammonia as nitrogenous sources, respectively, and the enzymatic activity was inhibited by purine 5′-ribonucleotide compounds and 5′, 5′″′-p 1, p 4-diguanosine tetraphosphate.

Orotate phosphoribosyltransferase from yeast: Studies of the structure of the pyrimidine substrate binding site

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate p K values were determined using computer fitting exercises. One such p K value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase ( K m = 75.5 ± 0.1 μM) leading to formation of an unstable nucleotide product.

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: nuclear magnetic relaxation studies of the structures of enzyme-bound phosphoribosyl 1-pyrophosphate

Nuclear magnetic relaxation rate measurements have been performed on the protons and phosphorus atoms of phosphoribosyl 1-pyrophosphate (PRibPP) in the presence and absence of paramagnetic chromium(III), cobalt(II), and manganese(II) ions. The longitudinal relaxation rates were then used to calculate interatomic distances between the magnetic nuclei and these paramagnetic probes, from which was devised a conformation of the PRibPP-metal ion complex in solution. Thereafter, the experiments were accomplished in the presence of Mn(II) and a series of orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) concentrations, and from these data were estimated the distances between Mn(II) and the PRibPP nuclei at the active sites of these two enzymes from yeast. Comparisons between the Mn(II)-PRibPP conformation in solution and this structure at the active sites of OPRTase and HGPRTase revealed that the metal ion remained coordinated with the pyrophosphate group of PRibPP in all instances, whereas the overall distances between the ribose ring and Mn(II) at the enzyme active sites were approximately 1 A further from the metal ion. Model building studies also revealed that the 5'-phosphate group of PRibPP is positioned directly over the ribose ring in solution and at the OPRTase and HGPRTase active sites and may protect the 1'-carbon of PRibPP against on-line displacements of pyrophosphate under these conditions, where the PRibPP-to-Mn(II) concentration ratio is greater than 2000.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.

Mode of action of 2-amino-6-chloro-1-deazapurine

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5′-phosphate which accumulated to concentrations as high as 1000 nmoles/10 9 cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibited the synthesis of 5′-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides. IMP dehydrogenase from L1210 cells was not inhibited by the nucleotides of 2-amino-6-chloropurine or 2-amino-6-chloro-1-deazapurine at 1 mM comcentrations. The nucleotide of 2-amino-6-chloropurine was dechlorinated by AMP deaminase; the apparent K m was about the same as that of AMP and the V max was 21% that of AMP. The nucleotide of 2-amino-6-chloro-1-deazapurine was not a substrate. Conversion of the nucleotide of 2-amino-6-chloropurine to GMP by the action of AMP deaminase explains both the lower toxicity of 2-amino-6-chloropurine as compared to its 1-deaza analog and its capacity to increase pools of guanine nucleotides. Thus, of the two compounds the 1-deaza analog may be the more interesting as an antitumor agent because of the greater metabolic stability of its nucleotide.

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.