PRPP Research Articles

Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.

Increase of PRPP enhances chemosensitivity of PRPS1 mutant acute lymphoblastic leukemia cells to 5‐Fluorouracil

Relapse‐specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate‐limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5‐Fluorouracil (5‐FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5‐FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5‐FU to FUMP and FUTP in Reh cells, to promote 5‐FU‐induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re‐targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse‐specific mutations of PRPS1 might benefit from 5‐FU‐based chemotherapy in clinical settings.

NAD metabolism and the SLC34 family: evidence for a liver-kidney axis regulating inorganic phosphate.

The solute carrier 34 (SLC34) family of membrane transporters is a major contributor to Pi homeostasis. Many factors are involved in regulating the SLC34 family. The roles of the bone mineral metabolism factors parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) in Pi homeostasis are well studied. Intracellular Pi is thought to be involved in energy metabolism, such as ATP production. Under certain conditions of altered energy metabolism, plasma Pi concentrations are affected by the regulation of a Pi shift into cells or release from the tissues. We recently investigated the mechanism of hepatectomy-related hypophosphatemia, which is thought to involve an unknown phosphaturic factor. Hepatectomy-related hypophosphatemia is due to impaired nicotinamide adenine dinucleotide (NAD) metabolism through its effects on the SLC34 family in the liver-kidney axis. The oxidized form of NAD, NAD+, is an essential cofactor in various cellular biochemical reactions. Levels of NAD+ and its reduced form NADH vary with the availability of dietary energy and nutrients. Nicotinamide phosphoribosyltransferase (Nampt) generates a key NAD+ intermediate, nicotinamide mononucleotide, from nicotinamide and 5-phosphoribosyl 1-pyrophosphate. The liver, an important organ of NAD metabolism, is thought to release metabolic products such as nicotinamide and may control NAD metabolism in other organs. Moreover, NAD is an important regulator of the circadian rhythm. Liver-specific Nampt-deficient mice and heterozygous Nampt mice have abnormal daily plasma Pi concentration oscillations. These data indicate that NAD metabolism in the intestine, liver, and kidney is closely related to Pi metabolism through the SLC34 family. Here, we review the relationship between the SLC34 family and NAD metabolism based on our recent studies.

Effect and Mechanism of Tanshinone I on the Radiosensitivity of Lung Cancer Cells.

Resistance to radiotherapy is one of the main obstacles to improving cancer prognoses. To effectively destroy cancer cells, novel radiation sensitizers are needed. Recently, several natural products have been shown to exhibit promising tumor-killing properties. However, little is known about the specific mechanisms of these natural compounds on cancer treatment. In this study, after screening a high-throughput natural product library, we identified tanshinone I (Tan I) as a potential radiation sensitizer in lung cancer cells. Lung cancer radioresistant cell lines, H358-IR and H157-IR, were first established to confirm the radioresistant phenotypes. After that, a natural product library was used to screen the potential radiation sensitizer. We further examined the inhibition functions of Tan I on radioresistant cancer cells via a series of experiments. Tan I significantly inhibited cell proliferation and clone formation, consequently enhancing radiosensitivity in radioresistant lung cancer cells, H358-IR and H157-IR. Stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics indicated that Tan I downregulates expression of pro-oncogenic protein phosphoribosyl pyrophosphate aminotransferase (PPAT) in both H358-IR and H157-IR cells. Further analysis of molecular docking showed that Tan I is well-docked into the active pocket of the structure of PPAT, serving as a potential PPAT inhibitor. Taken together, these findings suggest that inhibition of the tumor promoter PPAT by Tan I exerts marked inhibitory effects on radioresistant lung cancer cells, improving radiation efficacy.

Metabolic engineering of Escherichia coli for high-yield uridine production

Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3 g/L uridine and 4.5 g/L uracil in shake flask culture for 32 h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8 g/L. As carbamyl phosphate, aspartate, and 5′-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5 g/L. After 64 h of fed-batch fermentation, the final engineered strain UR6 produced 70.3 g/L uridine with a yield and productivity of 0.259 g/g glucose and 1.1 g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.

Proteomic Analysis of the Function of a Novel Cold-Regulated Multispanning Transmembrane Protein COR413-PM1 in Arabidopsis.

The plasma membrane is the first subcellular organ that senses low temperature, and it includes some spanning transmembrane proteins that play important roles in cold regulation. COR413-PM1 is a novel multispanning transmembrane cold-regulated protein; however, the related functions are not clear in Arabidopsis. We found the tolerance to freezing stress of cor413-pm1 was lower than wild-type (WT). A proteomics method was used to analyze the differentially abundant proteins (DAPs) between cor413-pm1 and WT. A total of 4143 protein groups were identified and 3139 were accurately quantitated. The DAPs associated with COR413-PM1 and freezing treatment were mainly involved in the metabolism of fatty acids, sugars, and purine. Quantitative real-time PCR (qRT-PCR) confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1 (FAB1) is involved in fatty acid metabolism and might affect the plasma membrane structure; fructokinase 3 (FRK3) and sucrose phosphate synthase A1 (SPSA1) play roles in sugar metabolism and may influence the ability of osmotic adjustment under freezing stress; and GLN phosphoribosyl pyrophosphate amidotransferase 2 (ASE2) affects freezing tolerance through purine metabolism pathways. In short, our results demonstrate that the multispanning transmembrane protein COR413-PM1 regulates plant tolerance to freezing stress by affecting the metabolism of fatty acids, sugars, and purine in Arabidopsis.

Dynamic metabolic response of Aspergillus niger to glucose perturbation: evidence of regulatory mechanism for reduced glucoamylase production

Environmental gradient is an important common issue during scale-up process for protein production. To address the dynamic regulatory mechanism of Aspergillus niger being exposed to inhomogeneous glucose concentrations, glucose perturbation were experimented on the steady state of A. niger chemostat culture, and dynamic profiles of the intracellular metabolites in central carbon metabolism were tracked in a time scale of seconds. The upper glycolysis and pentose phosphate pathway showed sharp variations after glucose perturbation, while the lower glycolysis, TCA cycle and amino acid pools represented a moderate and prolonged response due to the allosteric regulation of enzymes and buffering function of metabolites with large pool sizes. Improved glucose-6-phosphate enhanced the metabolic flux to PP pathway remarkably, which provided not only more redox cofactors (NADPH) for protein synthesis but also more precursors (phosphoribosyl pyrophosphate and ribose-5-phosphate) for cell growth. Moreover, reduction of the total adenine nucleotides and major precursor amino acids indicated the upregulated RNA synthesis was required to produce stress proteins, and partially explained the drop of glucoamylase production when A. niger experienced a fluctuated glucose concentration environment. These findings would be valuable for improving bioreactor operation, design, and scale-up from engineering or genetic aspects.

\u03b2-nicotinamide mononucleotide (NMN) production in Escherichia coli

Diabetes is a chronic and progressive disease with continuously increasing prevalence, rising financial pressure on the worldwide healthcare systems. Recently, the insulin resistance, hallmark of type 2 diabetes, was cured in mice treated with NAD+ precursor β-nicotinamide mononucleotide (NMN), no toxic effects being reported. However, NMN has a high price tag, more cost effective production methods are needed. This study proposes a biotechnological NMN production method in Escherichia coli. We show that bicistronic expression of recombinant nicotinamide phosphoribosyl transferase (Nampt) and phosphoribosyl pyrophosphate (PRPP) synthetase in the presence of nicotinamide (NAM) and lactose may be a successful strategy for cost effective NMN production. Protein expression vectors carrying NAMPT gene from Haemophilus ducreyi and PRPP synthetase from Bacillus amyloliquefaciens with L135I mutation were transformed in Escherichia coli BL21(DE3)pLysS. NMN production reached a maximum of 15.42 mg per L of bacterial culture (or 17.26 mg per gram of protein) in these cells grown in PYA8 medium supplemented with 0.1% NAM and 1% lactose.

Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.

Evolutionary convergence in the biosyntheses of the imidazole moieties of histidine and purines.

BackgroundThe imidazole group is an ubiquitous chemical motif present in several key types of biomolecules. It is a structural moiety of purines, and plays a central role in biological catalysis as part of the side-chain of histidine, the amino acid most frequently found in the catalytic site of enzymes. Histidine biosynthesis starts with both ATP and the pentose phosphoribosyl pyrophosphate (PRPP), which is also the precursor for the de novo synthesis of purines. These two anabolic pathways are also connected by the imidazole intermediate 5-aminoimidazole-4-carboxamide ribotide (AICAR), which is synthesized in both routes but used only in purine biosynthesis. Rather surprisingly, the imidazole moieties of histidine and purines are synthesized by different, non-homologous enzymes. As discussed here, this phenomenon can be understood as a case of functional molecular convergence.ResultsIn this work, we analyze these polyphyletic processes and argue that the independent origin of the corresponding enzymes is best explained by the differences in the function of each of the molecules to which the imidazole moiety is attached. Since the imidazole present in histidine is a catalytic moiety, its chemical arrangement allows it to act as an acid or a base. On the contrary, the de novo biosynthesis of purines starts with an activated ribose and all the successive intermediates are ribotides, with the key β-glycosidic bondage joining the ribose and the imidazole moiety. This prevents purine ribonucleotides to exhibit any imidazole-dependent catalytic activity, and may have been the critical trait for the evolution of two separate imidazole-synthesizing-enzymes. We also suggest that, in evolutionary terms, the biosynthesis of purines predated that of histidine.ConclusionsAs reviewed here, other biosynthetic routes for imidazole molecules are also found in extant metabolism, including the autocatalytic cyclization that occurs during the formation of creatinine from creatine phosphate, as well as the internal cyclization of the Ala-Ser-Gly motif of some members of the ammonia-lyase and aminomutase families, that lead to the MIO cofactor. The diversity of imidazole-synthesizing pathways highlights the biological significance of this key chemical group, whose biosyntheses evolved independently several times.

HAA1 and PRS3 overexpression boosts yeast tolerance towards acetic acid improving xylose or glucose consumption: unravelling the underlying mechanisms.

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes.

QM/MM analysis of effect of divalent metal ions on OPRT action

The role of Mg2+ cofactor in orotate phosphoribosyltransferase (OPRT) catalyzed synthesis of orotidine monophosphate (OMP) from phosphoribosyl pyrophosphate (PRPP) and orotate (OA) in substrate binding and the influence of the identity of the divalent metal ion on the reaction mechanism were addressed in this study using quantum mechanics/molecular mechanics framework. Energetics of migration and binding of different substrate complexes in the active site cavity was established. A quantitative analysis of various processes indicated the reaction pathway to consist of complexation of Mg2+ with PRPP, migration of Mg2+-PRPP and OA towards the active site, binding of OA to OPRT, and binding of Mg2+-PRPP complex to OA-OPRT complex. The mechanism of the reaction was unaltered by the change in the identity of divalent metal ion. Experimentally reported inhibiting character of Co2+ was explained on the basis of large Co2+-PRPP binding and migration energies. Mg2+, Ca2+, Mn2+, Co2+ and Zn2+ ions were screened computationally to assess their inhibiting/activating characteristics. Trends obtained by our computational investigations were in correspondence with experimentally reported trends.

Tandem riboswitches form a natural Boolean logic gate to control purine metabolism in bacteria.

Gene control systems sometimes interpret multiple signals to set the expression levels of the genes they regulate. In rare instances, ligand-binding riboswitch aptamers form tandem arrangements to approximate the function of specific two-input Boolean logic gates. Here, we report the discovery of riboswitch aptamers for phosphoribosyl pyrophosphate (PRPP) that naturally exist either in singlet arrangements, or occur in tandem with guanine aptamers. Tandem guanine-PRPP aptamers can bind the target ligands, either independently or in combination, to approximate the function expected for an IMPLY Boolean logic gate to regulate transcription of messenger RNAs for de novo purine biosynthesis in bacteria. The existence of sophisticated all-RNA regulatory systems that sense two ancient ribonucleotide derivatives to control synthesis of RNA molecules supports the hypothesis that RNA World organisms could have managed a complex metabolic state without the assistance of protein regulatory factors.

QM/MM reveals the sequence of substrate binding during OPRT action

Computational investigation of orotate phosphoribosyltransferase (OPRT) action, an enzymatic reaction between phosphoribosyl pyrophosphate (PRPP) and orotic acid (OA) to yield orotidine 5′-monophosphate (OMP), was carried out. Insights into the pathways of the substrate attack step of the reaction were developed under the quantum mechanics/molecular mechanics framework with S. cerevisiae strain as the representative enzyme bearer. Four pathways were proposed for PRPP and OA binding differing in the sequence of PRPP, OA and Mg2+ ion complexation with OPRT. The formation of Mg2+-OPRT complex was accompanied by a small energy change while the largest stabilization was observed for the formation of Mg2+-PRPP complex supporting the experimental observation of Mg2+-PRPP complex as the true substrate for the reaction. Formation of PRPP-OPRT complex was found to be energetically not probable rendering the pathway requiring Mg2+-OA complex not probable. Further, PRPP migration towards the active site was found to be energetically not favoured rendering the pathway involving Mg2+-OA complexation improbable. Migration of OA and Mg2+-PRPP complex towards the active site was found to be energetically probable with a large stabilization of the system when Mg2+-PRPP complex bound to the OA-OPRT complex. This conclusively proved the sequential binding of OA and Mg2+-PRPP complexes during OPRT action.

High expression of PRPS1 induces an anti-apoptotic effect in B-ALL cell lines and predicts an adverse prognosis in Chinese children with B‑ALL

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is closely associated with a number of diseases; however, its influence in B-cell acute lymphoblastic leukemia (B-ALL) and the potential molecular mechanisms involved remain unclear. The present study aimed to evaluate the expression of PRPS1 in Chinese children with B-ALL and to investigate the mechanism of action of PRPS1 in this disease. A Cell Counting Kit-8 (CCK-8) assay was performed to examine the proliferation of B-ALL Sup-B15 and Raji cells, and flow cytometric analysis was conducted to determine the cell cycle distribution and rate of apoptosis. The mRNA and protein expression levels of PRPS1, MYC proto-oncogene, bHLH transcription factor, cyclin E1, B-cell lymphoma-2 (Bcl-2), cyclin dependent kinase 2 and caspase-3 were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Elevated PRPS1 expression was associated with a high-risk stratification and poor prognosis in patients with B-ALL. Furthermore, overexpression of PRPS1 accelerated the growth of and inhibited apoptosis in Sup-B15 and Raji cells as well as increasing the expression of Bcl-2 to induce an anti-apoptotic effect in B-ALL cell lines. The results of the present study indicate that PRPS1 regulates multiple processes in B-ALL and may be an attractive therapeutic target.

Conversion of PRPS Hexamer to Monomer by AMPK-Mediated Phosphorylation Inhibits Nucleotide Synthesis in Response to Energy Stress.

Tumors override energy stress to grow. However, how nucleotide synthesis is regulated under energy stress is unclear. We demonstrate here that glucose deprivation or hypoxia results in the AMPK-mediated phosphorylation of phosphoribosyl pyrophosphate synthetase 1 (PRPS1) S180 and PRPS2 S183, leading to conversion of PRPS hexamers to monomers and thereby inhibiting PRPS1/2 activity, nucleotide synthesis, and nicotinamide adenine dinucleotide (NAD) production. Knock-in of nonphosphorylatable PRPS1/2 mutants, which have uninhibited activity, in brain tumor cells under energy stress exhausts cellular ATP and NADPH and increases reactive oxygen species levels, thereby promoting cell apoptosis. The expression of those mutants inhibits brain tumor formation and enhances the inhibitory effect of the glycolysis inhibitor 2-deoxy-d-glucose on tumor growth. Our findings highlight the significance of recalibrating tumor cell metabolism by fine-tuning nucleotide and NAD synthesis in tumor growth.Significance: Our findings elucidate an instrumental function of AMPK in direct regulation of nucleic acid and NAD synthesis in tumor cells in response to energy stress. AMPK phosphorylates PRPS1/2, converts PRPS1/2 hexamers to monomers, and inhibits PRPS1/2 activity and subsequent nucleotide and NAD synthesis to maintain tumor cell growth and survival. Cancer Discov; 8(1); 94-107. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

Inhibition of 3,5,2',4'-Tetrahydroxychalcone on Production of Uric Acid in Hypoxanthine-Induced Hyperuricemic Mice.

The mechanism of 3,5,2',4'-tetrahydroxychalcone on lowing urate level is still unknown. Here we investigated the effects of 3,5,2',4'-tetrahydroxychalcone on urate levels, xanthine oxidase/xanthine dehydrogenase (XOD/XDH) activities in hypoxanthine-induced hyperuricemic mice, as well as the effects of 3,5,2',4'-tetrahydroxychalcone on the mRNA expression levels and content of phosphoribosyl pyrophosphate synthetase (PRPS), phosphoribosyl pyrophosphate amidotransferase (PRPPAT) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Our results demonstrated that 3,5,2',4'-tetrahydroxychalcone (1.0, 2.0, and 4.0 mg/kg) reduced the uric acid levels in serum of the hyperuricemic mice in dose- and time-dependent manners. The activities of XOD/XDH in serum and liver were also significantly inhibited by 3,5,2',4'-tetrahydroxychalcone; In addition, 3,5,2',4'-tetrahydroxychalcone decreased the mRNA expression of HGPRT in brain and content of PRPS and PRPPAT in liver. These findings demonstrated that 3,5,2',4'-tetrahydroxychalcone suppresses uric acid production by affecting the critical enzymes, XOD/XDH, PRPS, PRPPAT and HGPRT in purine nucleotide metabolism.

A profound computational study to prioritize the disease-causing mutations in PRPS1 gene.

Charcot-Marie-Tooth disease (CMT) is one of the most commonly inherited congenital neurological disorders, affecting approximately 1 in 2500 in the US. About 80 genes were found to be in association with CMT. The phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is an essential enzyme in the primary stage of de novo and salvage nucleotide synthesis. The mutations in the PRPS1 gene leads to X-linked Charcot-Marie-Tooth neuropathy type 5 (CMTX5), PRS super activity, Arts syndrome, X-linked deafness-1, breast cancer, and colorectal cancer. In the present study, we obtained 20 missense mutations from UniProt and dbSNP databases and applied series of comprehensive in silico prediction methods to assess the degree of pathogenicity and stability. In silico tools predicted four missense mutations (D52H, M115T, L152P, and D203H) to be potential disease causing mutations. We further subjected the four mutations along with native protein to 50ns molecular dynamics simulation (MDS) using Gromacs package. The resulting trajectory files were analyzed to understand the stability differences caused by the mutations. We used the Root Mean Square Deviation (RMSD), Radius of Gyration (Rg), solvent accessibility surface area (SASA), Covariance matrix, Principal Component Analysis (PCA), Free Energy Landscape (FEL), and secondary structure analysis to assess the structural changes in the protein upon mutation. Our study suggests that the four mutations might affect the PRPS1 protein function and stability of the structure. The proposed study may serve as a platform for drug repositioning and personalized medicine for diseases that are caused by the PRPS1 deficiency.

Transition State Analysis of Adenosine Triphosphate Phosphoribosyltransferase.

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a DN*AN‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.