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.

Four immunoglobulin isotypes and IgD splice variants in urodele amphibians.

Until recently, different families of urodele amphibians were thought to express distinct subsets of immunoglobulin (Ig) isotypes. In this study, we explored cDNAs encoding Ig heavy-chains (H-chains) in three species of urodele amphibians. We found that Cynops pyrrhogaster, Pleurodeles waltl, and Ambystoma mexicanum each carry genes encoding four Ig H-chain isotypes, including IgM, IgY, IgD, and IgX, similar to those found in anuran amphibians. We also found that urodele IgDs have a long constant region similar to those found in anuran, reptiles, and bony fishes. We also found several putative IgD splice variants. Our findings indicated that P. waltl IgP is not a novel isotype but an IgD splice variant. Altogether, our findings indicate that IgD splice variants may be universally expressed among amphibian species.