Structure and Reaction Mechanism of YcjR, an Epimerase That Facilitates the Interconversion of d-Gulosides to d-Glucosides in Escherichia coli

Abstract

YcjR from Escherichia coli K-12 MG1655 catalyzes the manganese-dependent reversible epimerization of 3-keto-α-D-gulosides to the corresponding 3-keto-α-D-glucosides as a part of a proposed catabolic pathway for the transformation of D-gulosides to Dglucosides. 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 α-methyl-3-keto-D-glucoside at C4. However, the E240Q mutant can catalyze hydrogen−deuterium exchange of the proton at C4 of α-methyl-3-keto-D-glucoside in solvent D 2 O. 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.

Molecular Basis of Absorption at 340 nm of 3-Ketoglucosides under Alkaline Conditions

“…3-Ketosucrose 1, the oxidized product from sucrose, has been considered to be a building block for chemicals in organic synthesis [6,7]. Escherichia coli possesses a gene cluster consisting of those coding two NAD-dependent dehydrogenases oxidizing glucosides and gulosides at their C3 hydroxy groups, respectively, and a sugar isomerase interconverted the oxidized products [8,9]. Several bacteria metabolize 1,6-anhydro-β-D-glucopyranose (levoglucosan) 2 after oxidizing the C3 hydroxy group by NAD-dependent levoglucosan dehydrogenase (EC 1.1.1.425) to generate 3-ketolevoglucosan (1,6-anhydro-β-D-ribo-hexopyranos-3-ulose) 3 [10 12], followed by enzymatic intramolecular β-elimination [13].…”