Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes

Abstract

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-D-Galp-(1!3)-L-Ara and β-L-Arap-(1!3)-L-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-L-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-L-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-L-Rhap-(1!4)-β-D-Glcpα-(1!6)-β-D-Galp-(1!6)-D-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-β1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria.