Phosphoribosyl Diphosphate Research Articles

Interactions of Saccharomyces cerevisiae and Lactiplantibacillus plantarum Isolated from Light-Flavor Jiupei at Various Fermentation Temperatures.

Chinese Baijiu is a famous fermented alcoholic beverage in China. Interactions between key microorganisms, i.e., Saccharomyces cerevisiae and Lactiplantibacillus plantarum, have recently been reported at specific temperatures. However, empirical evidence of their interactions at various temperatures during fermentation is lacking. The results of this study demonstrated that S. cerevisiae significantly suppressed the viability and lactic acid yield of L. plantarum when they were cocultured above 15 °C. On the other hand, L. plantarum had no pronounced effect on the growth and ethanol yield of S. cerevisiae in coculture systems. S. cerevisiae was the main reducing sugar consumer. Inhibition of lactic acid production was also observed when elevated cell density of L. plantarum was introduced into the coculture system. A proteomic analysis indicated that the enzymes involved in glycolysis, lactate dehydrogenase, and proteins related to phosphoribosyl diphosphate, ribosome, and aminoacyl-tRNA biosynthesis in L. plantarum were less abundant in the coculture system. Collectively, our data demonstrated the antagonistic effect of S. cerevisiae on L. plantarum and provided insights for effective process management in light-flavor Baijiu fermentation.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Copper ions inhibit pentose phosphate pathway function in Staphylococcus aureus.

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.

Azotobacter chroococcum and Pseudomonas putida enhance pyrroloquinazoline alkaloids accumulation in Adhatoda vasica hairy roots by biotization

Adhatoda vasica is used in the treatment of cold, cough, chronic bronchitis, asthma, diarrhea, and dysentery. The biological activities of this species are attributed with the presence of alkaloids, triterpenoids, and flavonoids. Agrobacterium rhizogenes-mediated transformation of A. vasica, produces pyrroloquinazoline alkaloids, was achieved by infecting leaf discs with strain ATCC15834. The bacterial strain infected 82.7% leaf discs and 5–7 hairy root initials were developed from the cut edges of leaf discs. In this study, seven strains of Azotobacter chroococcum and five strains of Pseudomonas putida were used for the biotization of hairy roots. Plant growth-promoting rhizobacteria (PGPR) develops symbiotic association with roots of plants and increases the growth parameters of plants. PGPR (A. chroococcum and P. putida) increased the profiles of nitrogenase and acid phosphatase enzymes, biomass, dry matter contents, anthranilate synthase activity and accumulation of pyrroloquizoline alkaloids in the biotized hairy roots. Both enzymes (nitrogenase and acid phosphatase) maintain sufficient supply of nitrogen and dissolved phosphorus to the cells of hairy roots therefore, the levels of anthranilate synthase activity and pyrroloquinazoline alkaloids are increased. Total seven pyrroloquinazoline alkaloids (vasicine, vasicinone, vasicine acetate, 2-acetyl benzyl amine, vasicinolone, deoxyvasicine and vasicol) were identified from the biotized hairy roots of A. vasica. In our study, biotization increased the profiles of pyrroloquinazoline alkaloids therefore, this strategy may be used in increasing the production of medicinally important secondary metabolites in other plant species also. Our hypothetical model demonstrates that P. putida cell surface receptors receive root exudates by attaching on hairy roots. After attachment, the bacterial strain penetrates in the biotized hairy roots. This endophytic interaction stimulates acid phosphatase activity in the cells of biotized hairy roots. The P. putida plasmid gene (ppp1) expression led to the synthesis of acid phosphatase in cytosol. The enzyme enhances phosphorus availability as well as induces the formation of phosphoribosyl diphosphate. Later, phosphoribosyl diphosphate metabolizes to tryptophan and finally tryptophan converts to anthranilic acid. The synthesized anthranilic acid used in the synthesis of alkaloids in A. vasica.

Biological synthesis of nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-β-D-ribofuranoside 5'-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity.

Overexpression of Phosphoribosyl Pyrophosphate Synthase Enhances Resistance of Chlamydomonas to Ionizing Radiation.

The enzyme phosphoribosyl pyrophosphate synthase (PRPS) catalyzes the conversion of ribose 5-phosphate into phosphoribosyl diphosphate; the latter is a precursor of purine and pyrimidine nucleotides. Here, we investigated the function of PRPS from the single-celled green alga Chlamydomonas reinhardtii in its response to DNA damage from gamma radiation or the alkylating agent LiCl. CrPRPS transcripts were upregulated in cells treated with these agents. We generated CrPRPS-overexpressing transgenic lines to study the function of CrPRPS. When grown in culture with LiCl or exposed to gamma radiation, the transgenic cells grew faster and had a greater survival rate than wild-type cells. CrPRPS overexpression enhanced expression of genes associated with DNA damage response, namely RAD51, RAD1, and LIG1. We observed, from transcriptome analysis, upregulation of genes that code for key enzymes in purine metabolism, namely ribonucleoside-diphosphate pyrophosphokinase subunit M1, adenylate kinase, and nucleoside-diphosphate kinase. We conclude that CrPRPS may affect DNA repair process via regulation of de novo nucleotide synthesis.

Pharmacological Inhibition of HSP90 Radiosensitizes Head and Neck Squamous Cell Carcinoma Xenograft by Inhibition of DNA Damage Repair, Nucleotide Metabolism, and Radiation-Induced Tumor Vasculogenesis.

Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using liquid chromatography-Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5'-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow-derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.

Abstract 4024: NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD) biosynthesis. As NAMPT inhibitors (NAMPTi) have been shown to be substrates of NAMPT, binding of NAMPTi to NAMPT results in the NAMPTi-phosphoribose adduct (RP) that binds tightly and strongly inhibits the NAMPT enzyme. Structural analyses performed in this study unveiled the proximity of the NAMPTi-RP complex to the phosphoribosyl diphosphate (PRPP)-binding loop within the NAMPT catalytic site. The PRPP-binding loop of NAMPT is subject to conformational flexibility. The PRPP-binding loop domain is oriented to the outer side of the NAMPT dimer and can potentially serve as an antigen-binding site for antibodies. Interestingly, we show that the NAMPTi-RP adduct bound to NAMPT decreases NAMPT capture by an antibody that is directed against the c-terminal 5-phosphoribosyl-1-pyrophosphate-binding loop in NAMPT. This finding was then used to explore cellular NAMPT occupancy. The NAMPTi-RP complex displays a sustained, cellular NAMPT occupancy that correlates with the inhibition of NAMPT activity. Moreover, a good correlation between NAMPT occupancy, NAD decrease, and NAMPTi efficacy was observed in-vivo in a NCI-H82 small cell lung cancer (SCLC) xenograft model. NAMPT-occupancy assay can be used in clinical settings to better define the optimal dose levels and dose regimen for effective NAMPT inhibition. Citation Format: Maysoun Shomali, Sandrine Hilairet, Marianne Duhamel, Frederico Nardi, Thomas Bertrand, Geraldine Penarier, Laurent Debussche, Monsif Bouaboula. NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4024.

Systematic stress adaptation of Bacillus subtilis to tetracycline exposure.

To alleviate the harmful effects of antibiotics on the environment and human health, the stress response and molecular network of Bacillus under tetracycline stress were investigated using a proteomics approach. During the exposure process, Bacillus subtilis exhibited a strong adaptation mechanism. Cell membrane and intracellular reactive oxygen species (ROS) level returned to normal after 5 h. A total of 312 upregulated and 65 downregulated proteins were identified, mainly involved in metabolism and the synthesis of ribosomes, DNA, and RNA. After tetracycline exposure, the core metabolism network was accelerated to supply precursors for the synthesis of DNA, RNA, proteins, peptidoglycans, and saturated fatty acids that were involved in ribosome protection, and strengthened the cell wall and cell membrane. The signal transduction pathways involved were analyzed in association with the stress response of B. subtilis at 15 min of exposure to tetracycline. The primary damage to the ribosome by tetracycline activated a series of response proteins. Antitoxin and heat-shock proteins were activated for the global regulation of transcription and metabolism. Trigger factor Tig was upregulated to ensure proper initiation of transcription and aerobic respiration. Temperature-sensor protein VicR from the two-component system was used by the cell to regulate the composition of the cell wall and cell membrane. The over-consumption of metabolites, such as phosphoribosyl diphosphate (PRPP), purine nucleoside triphosphate (GTP), and acetyl-CoA forced the cells to assimilate more sugar for glycolysis. To this end, methyl-accepting chemotaxis proteins (MCPs) and sugar transportation protein PtsG were upregulated, simultaneously. Ultimately, peroxidase was activated to eliminate the redundant ROS, to minimize cell damage. These findings presented a system-level understanding of adaption processes of bacteria to antibiotic stress.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Novel Class III Phosphoribosyl Diphosphate Synthase: Structure and Properties of the Tetrameric, Phosphate-activated, Non-allosterically Inhibited Enzyme from Methanocaldococcus jannaschii

The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K(m) values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V(max) value as 2.20 mmol (minxmg of protein)(-1). ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M.jannaschii enzyme. The M.jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B.subtilis PRPP synthase. Soaking of the crystals with 5'-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases.

Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase

Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype enzyme. Kinetic characterization showed that the V(max) values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced, respectively, compared to the wildtype enzyme. The K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.

Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit the B. caldolyticus enzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP. Ki values are determined as 113 and 490 microm for ADP and GDP, respectively. At low concentrations ADP inhibition is linearly competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure.

Heterooligomeric Phosphoribosyl Diphosphate Synthase of Saccharomyces cerevisiae: COMBINATORIAL EXPRESSION OF THE FIVE PRS GENES IN ESCHERICHIA COLI

The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.

Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways.

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.

Class II Recombinant Phosphoribosyl Diphosphate Synthase from Spinach: PHOSPHATE INDEPENDENCE AND DIPHOSPHORYL DONOR SPECIFICITY

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.

Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of P i. Amino acid sequence comparison and phylogenetic analysis indicate that these two isozymes belong to a novel class of phosphoribosyl diphosphate synthases.

Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach.

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Deltaprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%-25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.

A reciprocal allosteric mechanism for efficient transfer of labile intermediates between active sites in CAD, the mammalian pyrimidine-biosynthetic multienzyme polypeptide.

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5'-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.

Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase.

The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP and (+)-1-alpha,2-alpha,3-alpha-trihydroxy-4-beta-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg2+ and the substrates were determined to occur via a steady state ordered mechanism in which Mg2+ binds to the enzyme first and ribose 5-phosphate binds last. Mg2+ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg x ATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate. Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by Mg x ADP indicated that Mg x ADP binding to the allosteric site may occur in competition with enzyme bound Mg2+. Ligand binding studies showed that 1 mol of Mg x ATP was bound per mol of phosphoribosyl diphosphate synthetase subunit, which indicated that the allosteric sites of the multimeric enzyme were not made up by inactive catalytic sites.