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AbstractIn 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‐(1→3)‐β‐l‐Araf‐(1→2)‐β‐l‐Araf‐(1→2)‐β‐l‐Araf, l‐Araf4), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide‐degrading enzymes. The l‐Araf4 motif was cleaved by GH43 α‐l‐arabinofuranosidase (Arafase) and converted to an l‐Araf3‐linked structure. The latter is then cleaved by GH121 β‐l‐arabinobiosidase (HypBA2), producing β‐l‐Araf‐(1→2)‐l‐Ara (β‐l‐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‐Araf1‐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‐Arafn) (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‐Araf1) motif.