Kinetic Studies of the Uracil Phosphoribosyltransferase Reaction Catalyzed by the Bacillus subtilis Pyrimidine Attenuation Regulatory Protein PyrR

PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60°C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg 2؉ , bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg 2؉ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.

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“…2). These values are quite similar to those observed with B. subtilis PyrR, which were 27 M and 0.56 mol per mol of PyrR, respectively (9).…”

Section: Resultssupporting

confidence: 76%

“…32 P]UMP and characterization of its binding to PyrR followed the procedure described previously (9). Dialysis was in 100 mM Tris-acetate, 10 mM K acetate, and 20% glycerol (pH 7.5) at 0°C for 40 to 48 h. Equimolar MgCl 2 and UMP were used.…”

Section: Characterization Of Ump Binding To Pyrr By Equilibrium Dialymentioning

confidence: 99%

Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus , Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

et al. 2005

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“…The kinetics of a ping‐pong reaction (i.e. parallel lines in double reciprocal plots of initial velocities vs. substrate concentration) was also observed for the PyrR protein (a regulatory RNA binding protein that also is a UPRTase) by Grabner and Switzer [34]. These authors attributed the kinetic behavior to an essentially irreversible conformational change (E‐PRPP to E′‐PRPP) of that enzyme in response to the binding of PRPP and preceding the binding of uracil.…”

Section: Reaction Mechanismmentioning

confidence: 84%

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

et al. 2005

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The phosphoribosyltransferases catalyze the formation of nucleotides by reaction with 5-phosphoribosyl-1-diphosphate (PRPP) and a nitrogenous base, releasing pyrophosphate as the second reaction product. The enzymes participate in the biosynthesis of the amino acids histidine and tryptophan, pyridine coenzymes, and all nucleotides by de novo synthesis and salvage pathways [1]. They are generally unregulated enzymes obeying hyperbolic saturation kinetics for the substrates, but there are exceptions. The glutamine PRPP amidotransferase, which catalyses the first step specific for de novo purine nucleotide synthesis, is feedback inhibited by AMP and GMP, and uracil phosphoribosyltransferase (UPRTase; a salvage enzyme that makes UMP from uracil and PRPP) was shown to be activated by GTP in several organisms e.g. The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60°C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number k cat and raised the K m values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two-and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (K D equal to 2 and 0.5 lm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.Abbreviations IPTG, isopropyl thio-b-D-galactoside; GdHCl, guanidinium chloride; PP i , inorganic diphosphate or pyrophosphate; PRPP, 5-phosphoribosyl-1-adiphosphate; UPRTase, uracil phosphoribosyltransferase or UMP synthase (EC 2.4.2.9).

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“…Many examples of enzymes that do not fall into the strict classification of sequential or ping‐pong mechanisms, but lie somewhere in between these two systems, have been reported [51–57]. These studies show that it can be misleading to diagnose a kinetic mechanism on the basis of only initial velocity patterns, and recommend including additional experiments to confirm the mechanism.…”

Section: Discussionmentioning

confidence: 99%

A novel prokaryotic l‐arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis

et al. 2010

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We report the first characterization of an l‐arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide‐derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well‐characterized human l‐arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an l‐arginine:glycine amidinotransferase by 1H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis–Menten kinetics in the presence of the non‐natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping‐pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (Tmax of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic l‐arginine:glycine amidinotransferases.

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Kinetic Studies of the Uracil Phosphoribosyltransferase Reaction Catalyzed by the Bacillus subtilis Pyrimidine Attenuation Regulatory Protein PyrR

Cited by 14 publications

References 35 publications

Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus , Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus , Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

A novel prokaryotic l‐arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis

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