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Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b)

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Title: Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b)
Authors: Okuyama, Masayuki Browse this author →KAKEN DB
Matsunaga, Kana Browse this author
Watanabe, Ken-ichi Browse this author
Yamashita, Keitaro Browse this author
Tagami, Takayoshi Browse this author
Kikuchi, Asako Browse this author
Ma, Min Browse this author
Klahan, Patcharapa Browse this author
Mori, Haruhide Browse this author →KAKEN DB
Yao, Min Browse this author →KAKEN DB
Kimura, Atsuo Browse this author →KAKEN DB
Keywords: carbohydrate synthesis
chemical rescue
crystal structure
enzyme kinetics
glycoside hydrolase
Issue Date: Mar-2017
Publisher: John Wiley & Sons
Journal Title: FEBS Journal
Volume: 284
Issue: 5
Start Page: 766
End Page: 783
Publisher DOI: 10.1111/febs.14018
PMID: 28103425
Abstract: The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from alpha-glycosidases can give poor yields of desired products because they require generally unstable beta-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 alpha-galactosidase, using more stable beta-galactosyl azide and alpha-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using beta-galactosyl azide and alpha-galactosyl transfer from alpha-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, beta-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, beta-lactosyl alpha-D-galactoside, was solved to elucidate the ligand-binding aspects of the alpha-galactosidase. Subtle differences at the beta ->alpha loops 1, 2 and 3 of the catalytic TIM barrel of the alpha-galactosidase from those of a homologous GH97 alpha-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in beta ->alpha loop 1 have separate roles. Trp312 of the alpha-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the alpha-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of alpha-galactoside recognition is conserved among GH27, 31, 36 and 97 alpha-galactosidases.
Rights: This is the peer reviewed version of the following article: FEBS Journal 284(5), pp766-783, 2017, which has been published in final form at 10.1111/febs.14018. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Type: article (author version)
URI: http://hdl.handle.net/2115/68390
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 木村 淳夫

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