Open AccessStructure and Reaction Mechanism of YcjR, an Epimerase That Facilitates the Interconversion of d -Gulosides to d -Glucosides in Escherichia coli
Author(s) -
Mark Mabanglo,
Jamison P. Huddleston,
Krishanu Mukherjee,
Zane W. Taylor,
Frank M. Raushel
Publication year - 2020
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00334
Subject(s) - chemistry , active site , protonation , stereochemistry , isomerization , deprotonation , divalent , manganese , escherichia coli , enzyme , catalysis , biochemistry , ion , organic chemistry , gene
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 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 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.