Functional Characterization of the ycjQRS Gene Cluster from Escherichia coli: A Novel Pathway for the Transformation of d-Gulosides to d-Glucosides.

A combination of bioinformatics, steady-state kinetics, and NMR spectroscopy has revealed the catalytic functions of YcjQ, YcjS, and YcjR from the ycj gene cluster in Escherichia coli K-12. YcjS was determined to be a 3-keto-d-glucoside dehydrogenase with a k_cat = 22 s^-1 and k_cat/ K_m = 2.3 × 10⁴ M^-1 s^-1 for the reduction of methyl α-3-keto-d-glucopyranoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD⁺-dependent oxidation of d-glucose, methyl β-d-glucopyranoside, and 1,5-anhydro-d-glucitol. YcjQ was determined to be a 3-keto-d-guloside dehydrogenase with k_cat = 18 s^-1 and k_cat/ K_m = 2.0 × 10³ M^-1 s^-1 for the reduction of methyl α-3-keto-gulopyranoside. This is the first reported dehydrogenase for the oxidation of d-gulose. YcjQ also exhibited catalytic activity with d-gulose and methyl β-d-gulopyranoside. The 3-keto products from both dehydrogenases were found to be extremely labile under alkaline conditions. The function of YcjR was demonstrated to be a C4 epimerase that interconverts 3-keto-d-gulopyranosides to 3-keto-d-glucopyranosides. These three enzymes, YcjQ, YcjR, and YcjS, thus constitute a previously unrecognized metabolic pathway for the transformation of d-gulosides to d-glucosides via the intermediate formation of 3-keto-d-guloside and 3-keto-d-glucoside.

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