Evolution of enzymatic activities in the enolase superfamily: crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis

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 R-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 mutan