Assimilation of arabinogalactan side chains with novel 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase in Bifidobacterium pseudocatenulatum

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum (B. longum) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289_0425) was characterized. Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289_0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on β-L-arabinopyranosyl disaccharide and larch AGP was examined. Results: MCC10289_0425 was identified to be an unprecedented 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn(119) to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth. Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures.