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A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron

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Title: A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron
Authors: Kikuchi, Asako Browse this author
Okuyama, Masayuki Browse this author →KAKEN DB
Kato, Koji Browse this author
Osaki, Shohei Browse this author
Ma, Min Browse this author
Kumagai, Yuya Browse this author
Matsunaga, Kana Browse this author
Klahan, Patcharapa Browse this author
Tagami, Takayoshi Browse this author
Yao, Min Browse this author →KAKEN DB
Kimura, Atsuo Browse this author →KAKEN DB
Keywords: Glycoside hydrolase family 97
Bacteroides thetaiotaomicron
Crystal structure
Substrate recognition
Issue Date: Nov-2017
Publisher: Elsevier
Journal Title: Biochimie
Volume: 142
Start Page: 41
End Page: 50
Publisher DOI: 10.1016/j.biochi.2017.08.003
PMID: 28804002
Abstract: Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, alpha-glucoside hydrolase and alpha-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 alpha-glucoside hydrolase and alpha-galactosidase, were characterized in this study. BT3664 was identified to be an alpha-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both beta-L-arabinopyranoside and alpha-D-galactopyranoside, and thus we designate BT3661 as a beta-L-arabinopyranosidase/alpha-D-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 alpha-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears G1u361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The kcat/Km value for the hydrolysis of p-nitrophenyl alpha-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl beta-L-arabinopyranoside. (C) 2017 Published by Elsevier B.V.
Rights: © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
Type: article (author version)
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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