Three asparagine synthetase genes of Bacillus subtilis.
(1/39)
Three asparagine synthetase genes, asnB, asnH, and asnO (yisO), were predicted from the sequence of the Bacillus subtilis genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of asnB was detected only during exponential growth, that of asnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnO was seen only later in sporulation. In a minimal medium, both asnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of asnH was partially repressed. Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprising yxbB, yxbA, yxnB, asnH, and yxaM. The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an Escherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In B. subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of asnB led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B. subtilis might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation. (+info)
X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution.
(2/39)
BACKGROUND: The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP. Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen. AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i). RESULTS: The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit. The final model of PurM consists of two crystallographically independent dimers and four sulfates. The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4%. The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers. A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved. CONCLUSIONS: The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains. (+info)
A highly conserved mechanism of regulated ribosome stalling mediated by fungal arginine attenuator peptides that appears independent of the charging status of arginyl-tRNAs.
(3/39)
The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control. It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S. cerevisiae, N. crassa, and wheat germ. Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system. The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC. Mutant AAPs lacking function in vivo did not function in vitro. Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems. Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg. The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations. Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA. (+info)
The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis.
(4/39)
The yexA gene encodes an 84 amino acid reading frame; in Bacillus subtilis it is positioned between the purC and purQ genes of the purine biosynthetic operon. Disruption of yexA resulted in a purine-auxotrophic phenotype. When yexA was expressed in trans it was able to complement a yexA mutation. Growth experiments and enzyme analysis of yexA mutant strains revealed a defective phosphoribosylformylglycinamidine synthetase (FGAM synthetase). In the organisms in which FGAM synthetase has been studied a single polypeptide is responsible for activity. In some organisms two separate genes - in B. subtilis the purL and purQ genes - encode polypeptides with similarity to the N-terminal and the C-terminal region, respectively, of the single-polypeptide FGAM synthetase. Thus, active FGAM synthetase in B. subtilis requires the yexA gene product in addition to the purL and purQ gene products. Open reading frames with sequence similarity to yexA are found in other Gram-positive organisms, in a cyanobacterium and in methanogenic archaea. The designation purS is proposed for this novel function in purine biosynthesis in B. subtilis. (+info)
Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation.
(5/39)
Neurospora crassa arg-2 mRNA contains an evolutionarily conserved upstream open reading frame (uORF) encoding the Arg attenuator peptide (AAP) that confers negative translational regulation in response to Arg. We examined the regulatory role of the AAP and the RNA encoding it using an N. crassa cell-free translation system. AAPs encoded by uORFs in four fungal mRNAs each conferred negative regulation in response to Arg by causing ribosome stalling at the uORF termination codon. Deleting the AAP non-conserved N terminus did not impair regulation, but deletions extending into the conserved region eliminated it. Introducing many silent mutations into a functional AAP coding region did not eliminate regulation, but a single additional nucleotide change altering the conserved AAP sequence abolished regulation. Therefore, the conserved peptide sequence, but not the mRNA sequence, appeared responsible for regulation. AAP extension at its C terminus resulted in Arg-mediated ribosomal stalling during translational elongation within the extended region and during termination. Comparison of Arg-mediated stalling at a rare or common codon revealed more stalling at the rare codon. These data indicate that the highly evolutionarily conserved peptide core functions within the ribosome to cause stalling; translational events at a potential stall site can influence the extent of stalling there. (+info)
BACKGROUND: Endogenous production of nitric oxide is vital for the decrease in pulmonary vascular resistance that normally occurs after birth. The precursor of nitric oxide is arginine, a urea-cycle intermediate. We hypothesized that low concentrations of arginine would correlate with the presence of persistent pulmonary hypertension in newborns and that the supply of this precursor would be affected by a functional polymorphism (the substitution of asparagine for threonine at position 1405 [T1405N]) in carbamoyl-phosphate synthetase, which controls the rate-limiting step of the urea cycle. METHODS: Plasma concentrations of amino acids and genotypes of the carbamoyl-phosphate synthetase variants were determined in 65 near-term neonates with respiratory distress. Plasma nitric oxide metabolites were measured in a subgroup of 10 patients. The results in infants with pulmonary hypertension, as assessed by echocardiography, were compared with those in infants without pulmonary hypertension. The frequencies of the carbamoyl-phosphate synthetase genotypes in the study population were assessed for Hardy-Weinberg equilibrium. RESULTS: As compared with infants without pulmonary hypertension, infants with pulmonary hypertension had lower mean (+/-SD) plasma concentrations of arginine (20.2+/-8.8 vs. 39.8+/-17.0 micromol per liter, P<0.001) and nitric oxide metabolites (18.8+/-12.7 vs. 47.2+/-11.2 micromol per liter, P=0.05). As compared with the general population, the infants in the study had a significantly skewed distribution of the genotypes for the carbamoyl-phosphate synthetase variants at position 1405 (P<0.005). None of the infants with pulmonary hypertension were homozygous for the T1405N polymorphism. CONCLUSIONS: Infants with persistent pulmonary hypertension have low plasma concentrations of arginine and nitric oxide metabolites. The simultaneous presence of diminished concentrations of precursors and breakdown products suggests that inadequate production of nitric oxide is involved in the pathogenesis of neonatal pulmonary hypertension. Our preliminary observations suggest that the genetically predetermined capacity of the urea cycle--in particular, the efficiency of carbamoyl-phosphate synthetase--may contribute to the availability of precursors for nitric oxide synthesis. (+info)
Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation.
(7/39)
Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Soll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838-12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organism's capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine. (+info)
Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia.
(8/39)
Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site. (+info)