Volume 24, Issue 5 e202200637
Research Article

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

Dr. Akihiro Ishiwata

Corresponding Author

Dr. Akihiro Ishiwata

RIKEN, Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan

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Hanako Tsunomachi

Hanako Tsunomachi

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima, 890-0065 Japan

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Kyohei Kameyama

Kyohei Kameyama

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima, 890-0065 Japan

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Dr. Kaeothip Sophon

Dr. Kaeothip Sophon

RIKEN, Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan

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Prof. Masayuki Nakamura

Prof. Masayuki Nakamura

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima, 890-0065 Japan

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Prof. Kanefumi Kitahara

Prof. Kanefumi Kitahara

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima, 890-0065 Japan

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Prof. Katsunori Tanaka

Prof. Katsunori Tanaka

RIKEN, Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan

Department of Chemical Science and Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo, 152-8552 Japan

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Prof. Yukishige Ito

Prof. Yukishige Ito

RIKEN, Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan

Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043 Japan

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Prof. Kiyotaka Fujita

Corresponding Author

Prof. Kiyotaka Fujita

Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima, 890-0065 Japan

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First published: 28 December 2022

Graphical Abstract

The distantly related homologue of GH127 Bll1HypBA1 β-l-arabinofuranosidase was recently identified as GH146 from Xanthomonas euvesicatoria along with paralogues from Bifidobacterium longum. One of them, Bll4HypBA1, can specifically degrade l-Araf1-Hyp in a similarly way to GH146 cysteine glycosidase, as was unequivocally revealed by monitoring the step-wise enzymatic cleavage of a synthetic extensin hydrophilic motif and potato lectin.

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-(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.

Conflict of interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.