Arginine-sensitive phenotype of mutations in pyrA of Salmonella typhimurium: role of ornithine carbamyltransferase in the assembly of mutant carbamylphosphate synthetase

Abstract

The phenotype of certain mutations in pyrA, the gene encoding carbamylphosphate synthetase (CPSase), is expressed only in the presence od exogenous arginine. In unsupplemented media, synthesis of carbamylphosphate and growth was almost normal; in arginine-containing media, synthesis of carbamylphosphate stopped, as did growth, as a consequence of starvation for pyrimidine. Genetic and biochemical evidence suggests that arginine exerts this inhibition by repressing the synthesis of ornithine carbamyltransferase (OTCase), the intracellular presence of which is required for assembly of the unequal subunits and proper functioning of the mutant CPSase. After the addition of arginine to a culture of the mutant, CPSase activity (glutamine dependent) characteristic of the intact holoenzyme progressively decreased, whereas activity (ammonia dependent) characteristic of the free large (alpha) subunit increased. Extracts of mutant cells contain free small (beta) subunits, as demonstrated directly by in vitro complementation using purified alpha subunits from wild type. The mutant enzyme from cultures grown in the presence of arginine had a markedly decreased affinity for adenosine 5'-triphosphate. Mutations in argR that cause depressed synthesis of OTCase suppressed the phenotype, and a certain mutation in argI, the gene encoding OTCase, enhanced it. In vitro experiments using purified enzyme confirm the stimulatory effect of OTCase on the activity of mutant CPSase.

Biosynthesis and metabolism of arginine in bacteria.

“…ARS and AUX pyrA mutants of S. typhimurium have been studied in some detail. It has been proposed that the formation of a complex between OTCase and CPSase is required for correct subunit assembly of the latter enzyme in the ARS mutants but not in wild-type strains (9). The AUX phenotype is often accompanied by cold sensitivity.…”

Arginine Auxotrophic Phenotype of Mutations in pyrA of Salmonella typhimurium : Role of N -Acetylornithine in the Maturation of Mutant Carbamylphosphate Synthetase

“…DISCUSSION Although complete loss of CPSase activity causes auxotrophy for both arginine and pyrimidine, missense mutations in pyrA (the gene encoding this enzyme) can lead to a variety of unexpected phenotypes. The biochemical basis ofone ofthese, the arginine-sensitive phenotype, has been described recently (5). In this paper, we have presented evidence relating to the biochemical nature of the arginine-auxotrophic (AUX) phenotype.…”