The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond.

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.