Structure and Reaction Mechanism of YcjR, an Epimerase that Facilitates the Interconversion of D-Gulosides to D-Glucosides in Escherichia coli.

YcjR from Escherichia coli K-12 MG1655 catalyzes the manganese-dependent reversible epimerization of 3-keto-alpha-D-gulosides to the corresponding 3-keto-alpha-D-glucosides as a part of a proposed catabolic pathway for the transformation of D-gulosides to D-glucosides. The three-dimensional structure of the manganese-bound enzyme was determined by X-ray crystallography. The divalent manganese ion is coordinated to the enzyme by ligation to Glu-146, Asp-179, His-205, and Glu-240. When either of the two active site glutamate residues is mutated to glutamine, the enzyme loses all catalytic activity for the epimerization of alpha-methyl-3-keto-D-glucoside at C4. However, the E240Qmutant can catalyze hydrogen-deuterium exchange of the proton at C4 of alpha-methyl-3-keto-D-glucoside in solvent D2O. The E146Q mutant does not catalyze this exchange reaction. These results indicate that YcjR catalyzes the isomerization of 3-keto-D-glucosides via proton abstraction at C4 by Glu-146 to form a cis-enediolate intermediate that is subsequently protonated on the opposite face by Glu-240 to generate the corresponding 3-keto-D-guloside. This conclusion is supported by docking of the cis-enediolate intermediate into the active site of YcjR based on the known binding orientation of D-fructose and D-psicose in the active site of D-psicose-3-epimerase.