Analysis of the mechanisms for uronate isomerase from E. coli, cobyrinic acid a,c-diamide synthetase from S. typhimurium, and cobyric acid synthetase from S. typhimurium.
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Uronate isomerase catalyzes the isomerization of D-glucuronate and Dfructuronate. This enzyme has been classified as a member of the amidohydrolase superfamily. The reaction catalyzed by uronate isomerase is analogous to the isomerization of aldose/ketose sugars. These interconversions can occur via two mechanisms, a hydride or proton transfer. The solvent exchange experiments and the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate catalyzed by the enzyme support a proton transfer. Assignment of the transferred proton as the proR proton further supports a proton transfer mechanism via a cis-enediol intermediate for uronate isomerase from E. coli. Cobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S. typhimurium catalyze ATP dependent amidations of carboxylate groups on the periphery of cobyrinic acid utilizing glutamine or ammonia as a nitrogen donor. The role of ATP in the reaction has been probed by positional isotope exchange (PIX). The results confirm the presence of phosphorylated intermediate species in the reactions catalyzed by cobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S. typhimurium. Cobyric acid synthetase catalyzes the amidation of carboxylate groups b, d, e, and g of adenosyl-cobyrinic acid a,c-diamide in the biosynthetic pathway for coenzyme B12. Analysis of the reaction time courses demonstrate the appearance of three unique intermediate species which are released from the active site after each amidation reaction. The identification of the intermediate species was accomplished by 1H, 15N HSQC NMR spectroscopy. The NMR spectrum of a sample quenched at the beginning of the reaction shows a single intermediate species corresponding to carboxamide e. Subsequent spectra establish the amidation order as e, d, b, and g. The structural basis for the dissociative and sequential reaction mechanism coupled with the rigid regiochemistry is unknown. However, mutations to aspartate 146 perturb the order of amidation. A NMR spectrum quenched early in the reaction with the D146N mutant shows two intermediate species corresponding to carboxamides e and d. Spectra of samples later in the reaction confirm the presence of multiple e, d, and g amide species. The reaction is completed with the amidation of carboxylate b.
cobyrinic acid a
Williams, LaKenya (2007). Analysis of the mechanisms for uronate isomerase from E. coli, cobyrinic acid a,c-diamide synthetase from S. typhimurium, and cobyric acid synthetase from S. typhimurium.. Doctoral dissertation, Texas A&M University. Available electronically from