3hk9 | pdb_00003hk9
Crystal structure of uronate isomerase from Bacillus halodurans complexed with zinc and D-Glucuronate
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedUronate 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 catalyzes primarily hydrolytic reactions. The chemical mechanism and active site structure of URI was investigated in an attempt to obtain a greater understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been re-forged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km 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 as 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/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 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. The Mechanism of the Reaction Catalyzed by Uronate Isomerase Illustrates How an Isomerase May Have Evolved from a Hydrolase within the Amidohydrolase Superfamily.,Nguyen TT, Fedorov AA, Williams L, Fedorov EV, Li Y, Xu C, Almo SC, Raushel FM Biochemistry. 2009 Aug 14. PMID:19678710[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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