Evolution of enzymatic activities in the enolase superfamily: Crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli

D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydra tion of both D-glucarate and L-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup o f the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the a-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 i n the active site of MR. Crystals of GlucD have been obtained into which th e 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 th ese complexes have been determined and reveal the identities of the ligands for the required Mg2+ (Asp(235), Glu(266), and Asn(289)) as well as confir m the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-g lucarate, 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 Ly(207) and His(339) are reversed f rom 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 L ys(207) and His(339) as was possible for the homologous Lys(166) and His(29 7) in the MR-catalyzed reaction.