Structure-function analysis and study on reaction mechanism of microbial cysteine glycosidases

2019 Fiscal Year Final Research Report

• Project/Area Number: 15H02443
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Fushinobu Shinya 東京大学, 大学院農学生命科学研究科(農学部), 教授 (00302589)
• Co-Investigator(Kenkyū-buntansha): 藤田 清貴 鹿児島大学, 農水産獣医学域農学系, 准教授 (20381189) ; 石渡 明弘 国立研究開発法人理化学研究所, 開拓研究本部, 専任研究員 (70342748)
• Project Period (FY): 2015-04-01 – 2019-03-31
• Keywords: 酵素 / タンパク質 / 有機化学 / 応用微生物学 / 糖鎖 / 糖質関連酵素 / 植物病原菌 / 選択的化学合成

Outline of Final Research Achievements

We found a novel type of glycoside hydrolase (beta-L-Araf-ase HypBA1) that has the Cys nucleophile coordinated to Zn. In this study, an examination of its reaction mechanism and structural analysis of related enzymes (proteins) were performed. Novel mechanism-based inhibitors that specifically react with the Cys residue were developed. Using these inhibitors, we could reveal the three-dimensional structures of all of the reaction steps of HypBA1. We also found novel beta-L-Araf-ases and performed functional and structural analyses with related enzymes. Characteristics of the enzymes and proteins involved in the beta-arabinooligosaccharide-degradation were revealed.

Free Research Field

酵素学、構造生物学

Academic Significance and Societal Importance of the Research Achievements

活性中心がCysである糖質加水分解酵素としてはこれ以外に全く前例がなく新規な発見である。さらにHypBA1で反応前（酵素-基質ミカエリス型複合体）、反応中間体アナログ（ハロアセトアミド体阻害剤との複合体）、反応後（産物であるβ-L-Arafとの複合体）の全てで立体構造に成功し、反応機構を解明した。本酵素は善玉菌として有名な腸内細菌であるビフィズス菌が食品に含まれるβ-アラビノオリゴ糖分解に関わる経路で重要な役割を担っており、ヒトの健康に有効なプレバイオティクス開発に繋がると期待される。