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

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 k cat = 22 s −1 , and k cat /K m = 2.3 × 10 4 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-Dglucopyranoside, 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 × 10 3 M −1 s −1 for the reduction of methyl α-3-keto-gulopyranoside. This is 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 C-4 epimerase that interconverts 3-keto-Dgulopyranosides 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 Dglucosides via the intermediate formation of 3-keto-D-guloside and 3-keto-D-glucoside.

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

“…Previously, we identified a cluster of 12 genes in Escherichia coli K-12 MG1655 (Figure ) that was predicted to encode enzymes of unknown function involved in the degradation of complex carbohydrates . We, and others, have successfully elucidated the catalytic function for several of these enzymes. − YcjT catalyzes the phosphorolysis of kojibiose (α-1,2- d -glucose- d -glucose) to d -glucose and β- d -glucose 1-phosphate . YcjU is a β-phosphoglucomutase that catalyzes the conversion of β- d -glucose 1-phosphate to glucose 6-phosphate.…”

“…YcjM is an α- d -glucosyl-2-glycerate phosphorylase that catalyzes the formation of d -glycerate and α-glucose 1-phosphate from α-1,2- d -glucose d -glycerate and phosphate. YcjQ is a dehydrogenase that catalyzes the NAD + -dependent oxidation of d -gulosides ( 1 ) at C3, forming the corresponding 3-keto- d -guloside ( 2 ) . YcjR catalyzes the isomerization of 2 at C4 to form 3-keto- d -glucoside ( 3 ).…”

“…YcjR catalyzes the isomerization of 2 at C4 to form 3-keto- d -glucoside ( 3 ). YcjS is a dehydrogenase that catalyzes the NADH-dependent reduction of 3-keto- d -glucoside ( 3 ) to d -glucoside ( 4 ) . Of the remaining genes in the cluster, five are annotated as sugar transporters (YcjN, YcjO, YcjP, YcjV, and OmpG), and YcjW is a LacI-type repressor. , The known chemical transformations are highlighted in Figure .…”

“…Of particular interest here is the enzyme YcjR. This enzyme is a member of cog1082 and has been shown to catalyze the epimerization of 3-keto-α- d -gulosides ( 2 ) to 3-keto-α- d -glucosides ( 3 ) by inversion of the stereochemistry at C4 . This enzyme thus enables the conversion of d -gulosides to the corresponding d -glucosides in conjunction with YcjQ and YcjS .…”

“…This enzyme is a member of cog1082 and has been shown to catalyze the epimerization of 3-keto-α- d -gulosides ( 2 ) to 3-keto-α- d -glucosides ( 3 ) by inversion of the stereochemistry at C4 . This enzyme thus enables the conversion of d -gulosides to the corresponding d -glucosides in conjunction with YcjQ and YcjS . Other functionally characterized members of cog1082 include d -psicose-3-epimerase, , d -tagatose-3-epimerase, and l -ribulose-3-epimerase .…”