Bifidobacterial GH146 β‐l‐Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline‐Rich Glycoproteins

Abstract

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-, whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf 4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf 3 -linked structure. The latter is then cleaved by GH121 β-larabinobiosidase (HypBA2), producing β-l-Araf-(1!2)-l-Ara (βl-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf 1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Araf n ) (n = 4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf 1 ) motif.