Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis.

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of ß-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl ß-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-ß-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl ß-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be ß-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-ß-d-glucopyranosyl ß-d-glucuronide structure released from bacterial and plant acidic carbohydrates.