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Structural insights reveal the second base catalyst of isomaltose glucohydrolase

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Title: Structural insights reveal the second base catalyst of isomaltose glucohydrolase
Authors: Tagami, Takayoshi Browse this author →KAKEN DB
Chen, Minghao Browse this author
Furunaga, Yuta Browse this author
Kikuchi, Asako Browse this author
Sadahiro, Juri Browse this author
Lang, Weeranuch Browse this author
Okuyama, Masayuki Browse this author →KAKEN DB
Tanaka, Yoshikazu Browse this author
Iwasaki, Tomohito Browse this author
Yao, Min Browse this author
Kimura, Atsuo Browse this author →KAKEN DB
Keywords: condensation
glycoside hydrolase
kinetic analysis
native SAD
pH-activity profile
Issue Date: 1-Feb-2023
Publisher: John Wiley & Sons
Journal Title: FEBS Journal
Volume: 289
Issue: 4
Start Page: 1118
End Page: 1134
Publisher DOI: 10.1111/febs.16237
Abstract: Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-k(cat) and pH-k(cat)/K-m profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a beta-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.
Rights: This is the peer reviewed version of the following article: Tagami, T., Chen, M., Furunaga, Y., Kikuchi, A., Sadahiro, J., Lang, W., Okuyama, M., Tanaka, Y., Iwasaki, T., Yao, M. and Kimura, A. (2022), Structural insights reveal the second base catalyst of isomaltose glucohydrolase. FEBS J, 289: 1118-1134, which has been published in final form at This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.
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: 田上 貴祥

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