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 kcat = 22 s-1, and kcat/Km = 2.3 x 104 M-1 s-1 for the reduction of α-methyl-3-keto-D-glucoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD+-dependent oxidation of D-glucose, β-methyl-D-glucoside, 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 x 103 M-1 s-1 for the reduction of α-methyl-3-keto-guloside. This is first reported dehydrogenase for D-gulose. YcjQ also exhibited catalytic activity with D-gulose and β-methyl-D-guloside. 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 C-4 epimerase that interconverts 3-keto-D-gulosides to 3-keto-D-glucosides. 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.