Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2.
Cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium (CbiA) is the first glutamine amidotransferase in the anaerobic biosynthetic pathway of vitamin B(12) and catalyzes the ATP-dependent synthesis of cobyrinic acid a,c-diamide from cobyrinic acid using either glutamine or ammonia as the nitrogen source. The cbiA gene was cloned, the overexpressed protein was purified to homogeneity, and the kinetic parameters were determined. CbiA is a monomer with K(m) values of 0.74, 2.7, 53, and 26 200 microM for cobyrinic acid, ATP, glutamine, and ammonia, respectively. Analysis of the glutaminase partial reaction demonstrated that the hydrolysis of glutamine and the synthesis of the cobyrinic acid a,c-diamide product are uncoupled. The time course for the synthesis of the diamide product and positional isotope exchange experiments demonstrate that CbiA catalyzes the sequential amidation of the c- and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate. These results support a model for the catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate. Several conserved residues in the synthetase active site were mutated to address the molecular basis of the amidation order; however, no changes in the order of amidation were obtained. The mutants D45N, D48N, and E90Q have a dramatic effect on the catalytic activity, whereas no effect was found for the mutant D97N. The substitutions by alanine of L47 and Y46 residues specifically decrease the affinity of the enzyme for the c-monoamide intermediate.
Department of Chemistry, Post Office Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
Vicente
V
Fresquet
LaKenya
L
Williams
Frank M
FM
Raushel
D019943:Amino Acid Substitution; D002384:Catalysis; D003001:Cloning, Molecular; D007700:Kinetics; D010103:Oxygen Isotopes; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010103:Oxygen Isotopes; D000637:Transaminases; C065829:cobyrinic acid a,c-diamide synthase