Dataset Information

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

ABSTRACT: Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.

SUBMITTER: Williams L

PROVIDER: S-EPMC2505117 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Williams LaKenya L Nguyen Tinh T Li Yingchun Y Porter Tamiko N TN Raushel Frank M FM

Biochemistry 20060601 24

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of ...[more]

PMID: 16768441

Similar Datasets

Project description:Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for k(cat) and k(cat)/K(m) for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. 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 from the end of beta-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed 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-355.

Dataset Information

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Publications

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets