Biochemical characterization of 1,5-anhydro-D-glucitol kinase from Clostridium symbiosum ATCC 14940

1,5-Anhydro-D-glucitol (1,5-AG), a 1-deoxy form of glucopyranose, is a major dietary-derived polyol in the human body and serves as a sensitive clinical marker for short-term glycemic control in diabetic patients. Its phosphorylated derivative, 1,5-anhydro-D-glucitol-6-phosphate (1,5-AG6P), is a non-canonical metabolite formed when glucose phosphorylating enzymes erroneously act on 1,5-AG. Accumulation of 1,5-AG6P is associated with neutrophil dysfunction. Recently, the Zhang group reported a gut bacterial pathway for 1,5-AG catabolism, in which the key enzyme is a glycyl radical enzyme (GRE) responsible for the ring-opening C−O cleavage of 1,5-AG6P. In this report, through a synergistic analysis of sequence similarity network (SSN) and genome neighborhood network (GNN) of this GRE homologs, we uncovered novel ATP-dependent 1,5-AG kinases belonging to the ROK family (PF00480). These enzymes exhibit higher catalytic efficiencies for 1,5-AG than for D-glucose, driven by both a smaller K M and a larger k cat . The substrate preference contrasts with that of other hexokinases and ADP-dependent glucokinases, which typically show a marked preference for D-glucose. This study expands the biocatalytic toolbox for the enzymatic synthesis of 1,5-AG6P, offering a more efficient and convenient approach, and reinforces the value of the “genomic enzymology” strategy applicable for enzyme function discovery.