Structure of the allosteric regulatory enzyme of purine biosynthesis.

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR.

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.

BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.

Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast.

In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells. In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed. Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source.

Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines.

The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role. A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described. The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance. Twelve such mutants were isolated and sequenced. All resulted from point mutations in the pyrR gene.

Non-redox roles for iron-sulfur clusters in enzymes.

In recent years a number of enzymes have been discovered which, contrary to prior expectations, contain FeS clusters but do not participate in redox reactions. In all cases but one, where the FeS cluster in these enzymes has been identified, it is a [4Fe-4S] cluster. In mammalian aconitase a single Fe atom of the [4Fe-4S] cluster participates in catalysis of hydration-dehydration reactions by direct ligation to the substrates. A number of hydrolyases containing FeS clusters have now been identified. In Bacillus subtilis glutamine phosphoribosyl-pyrophosphate amidotransferase the [4Fe-4S] cluster is essential for the active structure of the enzyme, but probably does not participate directly in catalysis. Rather, the cluster may serve as part of a mechanism of oxidative inactivation of the enzyme in vivo, which is followed by its intracellular degradation. The role played by a [4Fe-4S] cluster in Escherichia coli endonuclease III is at present completely unknown. Thus, a number of novel roles for FeS clusters in enzymology and protein structure have been discovered, and more novel findings must be anticipated.

Structure of the divalent cation.nucleotide complex at the active site of phosphoribosylpyrophosphate synthetase.

When Mg2+ was used as the activating cation, the phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) of Salmonella typhimurium showed absolute specificity for the A(S) enantiomer of adenosine 5'-O-(1-thiotriphosphate), which gave a Km of 72 +/- 10 microM and a Vmax of 111 +/- 5 mumol/min/mg. The corresponding values for ATP were 46 +/- 3 microM and approximately 107 mumol/min/mg. Under the same conditions the B(R) isomer was a linearly competitive inhibitor (Ki = 54 +/- 11 microM) with respect to ATP. When Cd2+ replaced Mg2+, the two isomers reacted at comparable rates (Vmax (A)/Vmax (B) approximately equal to 0.8). This change in specificity suggests that the alpha-phosphate of ATP is liganded to a divalent cation during catalysis. Adenosine 5'-O-thiomonophosphate was 34-fold more effective as a product inhibitor when Cd2+ replaced Mg2+, while the effectiveness of AMP was not altered. This result suggests a divalent cation bridge between the enzyme and the alpha-phosphate of nucleotides. The results of these and previously published experiments enable us to propose a structure and stereochemical configuration for the divalent cation.ATP complex at the active site of phosphoribosylpyrophosphate synthetase.

Characterization of pyrimidine-repressible and arginine-repressible carbamyl phosphate synthetases from Bacillus subtilis.

The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine.

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation.

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis.

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca2+ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca2+ ions and gramicidin S; its pH and temperature optima were 9.0 and 37 degrees C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.

Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB).

The Bacillus subtilis gene (pyrB), which encodes aspartate transcarbamylase (ATCase), was cloned on a HindIII restriction endonuclease fragment inserted into the pUC13 plasmid vector. B. subtilis pyrB was expressed in Escherichia coli, as judged by complementation of E. coli pyrB mutants and production of enzyme that was specifically inhibited by antibody directed against B. subtilis ATCase. The extent of expression was strongly dependent on the orientation of the inserted DNA in the vector, which suggested that transcription was initiated from vector-borne (rather than B. subtilis) promoters. The entire 1098-base pair HindIII fragment of B. subtilis DNA was sequenced by the Maxam-Gilbert method. The amino acid sequence of B. subtilis ATCase was deduced from a 305-codon open reading frame and agreed very well with analyses of the purified enzyme. Comparison of the sequence of B. subtilis ATCase with that of E. coli ATCase catalytic subunit, for which the three-dimensional structure is known, revealed many homologous residues of probable importance in catalysis and structural folding of ATCases. The significance of homology to E. coli ornithine transcarbamylases was also analyzed. The sequences of the 5' and 3' flanking regions to pyrB encode open reading frames in both cases which overlap with pyrB by eight and six codons, respectively. It is probable that these open reading frames encode other enzymes of a coordinately regulated unit. The sequence 5' to pyrB also encodes an mRNA bearing a pyrimidine-rich sequence followed by a typical sequence for a rho-independent transcription terminator. The presence of these elements and the 5' open reading frame suggest that B. subtilis pyrB, like E. coli pyrBI, is regulated by an attenuation mechanism.

Aspartokinase III, a new isozyme in Bacillus subtilis 168.

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.

Sulfhydryl chemistry of Salmonella typhimurium phosphoribosylpyrophosphate synthetase: identification of two classes of cysteinyl residues.

Sulfhydryl-specific reagents were used to study the reactivities and function of the four cysteinyl residues per subunit present in Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. In the presence of high concentrations of denaturants all four cysteinyl residues reacted with sulfhydryl-specific reagents. In the absence or in the presence of low levels of denaturing agents, two classes of cysteinyl residues were identified. A single sulfhydryl reacted rapidly with iodoacetamide and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) without significant loss of enzymatic activity. This single sulfhydryl was identified as Cys-229 by reaction with iodo[1-14C]acetamide, followed by isolation and sequence analysis of a single radiolabeled peptide. The three remaining sulfhydryls reacted to various extents depending on the conditions and sulfhydryl-specific reagents employed. At low Pi concentrations, these residues reacted fully with DTNB, leading to an 80 to 90% loss of enzymatic activity. ATP and high levels of Pi prevented this reaction. These results, along with studies comparing the S. typhimurium PRPP synthetase sequence with the sequences of PRPP synthetases from other species, suggest that the cysteinyl residues in the Salmonella enzyme are not catalytically essential. That one or more of the three less reactive residues may lie in or near the active site is not excluded.