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.

Evidence for substrate stabilization in regulation of the degradation of Bacillus subtilis aspartate transcarbamylase in vivo.

Aspartate transcarbamylase (ATCase) is rapidly degraded in Bacillus subtilis cells that are starved for a carbon or nitrogen source or a required amino acid. The hypothesis that ATCase degradation may be regulated in vivo by protection of the enzyme by substrate binding was tested by studies of a mutant ATCase (Arg99 to Ala, R99A), which binds substrate so poorly that it fails to support pyrimidine-independent growth in a pyrB strain, but still has 10% of normal activity when saturated with substrates. Unlike normal ATCase, R99A ATCase was degraded rapidly in exponentially growing cells. Degradation of the mutant enzyme was two-fold slower in a relA strain, as was degradation of the normal ATCase. The stability of purified R99A ATCase to denaturation by heat or guanidine hydrochloride was identical to that of wild-type ATCase, as was its circular dichroic spectrum. The wild-type and R99A ATCase were degraded identically in vitro by subtilisin, except that the mutant enzyme was much less effectively protected against cleavage by carbamyl phosphate, as expected. The carbamyl phosphate pool in glucose-limited B. subtilis cells was only one-third of the pool in exponentially growing cells. These results indicate that protection of ATCase by carbamyl phosphate binding could be one of the elements that regulate ATCase stability in vivo. However, carbamyl phosphate pools were the same in cells grown with ammonium ions and with a mixture of 20 common amino acids, conditions under which ATCase stability in vivo differs. Thus, other means of regulating ATCase degradation must also exist.

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.

The degA gene product accelerates degradation of Bacillus subtilis phosphoribosylpyrophosphate amidotransferase in Escherichia coli.

A search for genes involved in the inactivation and degradation of enzymes in sporulating Bacillus subtilis led to identification of the B. subtilis degA gene, whose product stimulates degradation of B. subtilis glutamine phosphoribosylpyrophosphate amidotransferase in Escherichia coli cells. degA encodes a 36.7-kDa protein that has sequence similarity to several E. coli and B. subtilis regulatory proteins of the LacI class. B. subtilis degA::cat insertional inactivation mutants had no detectable defect in the inactivation or degradation of phosphoribosylpyrophosphate amidotransferase in glucose- or lysine-starved B. subtilis cells, however. We suggest that degA encodes either a novel protease or, more likely, a gene that stimulates production of such a protease.

Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo.

The Bacillus subtilis pyr operon is regulated by a transcriptional attenuation mechanism that requires the PyrR regulatory protein. Multicopy plasmids that could be transcribed to yield segments of RNA from the attenuation regions of the pyr operon induced derepression of chromosomal pyr genes, whereas plasmids that could not yield pyr RNA did not. We conclude that pyr RNA acts by titrating the PyrR protein and preventing it from regulating pyr attenuation.

Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis.

An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis. The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII. We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.

Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro.

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).

Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo.

Glutamine phosphoribosylpyrophosphate amidotransferase is stable in growing cells, but is inactivated in an oxygen-dependent process at various rates in starving or antibiotic-treated cells. On the basis of studies of the purified enzyme, we suggested (D.A. Bernlohr and R.L. Switzer, Biochemistry 20:5675-5681, 1981) that the inactivation in vivo was regulated by substrate stabilization and a competition between stabilizing (AMP) and destabilizing (GMP, GDP, and ADP) nucleotides. This proposal was tested by measuring the intracellular levels of these metabolites under cultural conditions in which the stability of the amidotransferase varied. The results established that the stability of amidotransferase in vivo cannot be explained by the simple interactions observed in vitro. Metabolite levels associated with stability of the enzyme in growing cells did not confer stability under other conditions, such as ammonia starvation or refeeding of glucose-starved cells. The data suggest that a previously unrecognized event, possibly a covalent modification of amidotransferase, is required to mark the enzyme for oxygen-dependent inactivation.