Evolution of Enzymatic Activities in the Enolase Superfamily: Crystallographic and Mutagenesis Studies of the Reaction Catalyzed by d-Glucarate Dehydratase from Escherichia coli,

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Abstract
D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both d-glucarate and l-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the α-proton of a carboxylate anion substrate. Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR. Crystals of GlucD have been obtained into which the substrate d-glucarate and two competitive inhibitors, 4-deoxy-d-glucarate and xylarohydroxamate, could be diffused; d-glucarate is converted to the dehydration product, 5-keto-4-deoxy-d-glucarate (KDG). The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg2+ (Asp235, Glu266, and Asn289) as well as confirm the expected presence of Lys207 and His339, the catalytic bases that are properly positioned to abstract the proton from C5 of l-idarate and d-glucarate, respectively. Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg2+, with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys207 and His339 are reversed from the predictions made on the basis of the established structure−function relationships for the MR-catalyzed reaction. The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys207 and His339 as was possible for the homologous Lys166 and His297 in the MR-catalyzed reaction.