Functional Characterization of the ycjQRS Gene Cluster from Escherichia coli: A Novel Pathway for the Transformation of d-Gulosides to d-Glucosides.
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abstract
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 kcat = 22 s-1 and kcat/ Km = 2.3 104 M-1 s-1 for the reduction of methyl -3-keto-d-glucopyranoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD+-dependent oxidation of d-glucose, methyl -d-glucopyranoside, and 1,5-anhydro-d-glucitol. YcjQ was determined to be a 3-keto-d-guloside dehydrogenase with kcat = 18 s-1 and kcat/ Km = 2.0 103 M-1 s-1 for the reduction of methyl -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 -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.
