Division of Biological sciences, Graduate School of Science, Hokkaido University* Major of Sustainable Resource Science, Graduate School of Bioresources, Mie University, Japan** Major of Sustainable Resource Science, Graduate School of Bioresources, Mie University, Japan***
○Yu Kitago* Shuichi Karita** Nobuhisa Watanabe* Kazuo Sakka*** Isao Tanaka*
Cellulosome is the huge multiprotein complex that consists of the noncatalytic scaffolding protein and the various enzymatic proteins. The cellulosomal enzymatic proteins contain many kinds of cellulases such as endoglucanases, xylanases, mannanases, cellobiohydrolases, glucosidases, etc. This variety of enzymatic proteins makes it possible to degrade crystalline cellulose quite efficiently. In these enzymatic proteins, enzymes that belong to glycoside hydrolase family (GH family) 44 appear in some celulolytic bacteria's cellulosome, and those had been only known as its endoglucanase, lichenase, xylanase and xyloglucanase activity.
This study aims to reveal the detailed structure and mechanisms of the GH family 44 enzymes by X-ray crystallographic study of Cel44A from Clostridium thermocellum. Cel44A was overexpressed, purified and crystallized. The X-ray diffraction datasets of the wild type and the E186Q mutant complexed with various substrates were collected at SPring-8 and Photon Factory. The primary phases were solved by the MAD method using zinc ion, and the refinement procedure is performed at 0.96 Å resolution. The revealed structure shows that Cel44A consists of TIM-like barrel domain and b-sandwich domain, and suggests that two glutamic acid residues, Glu186 and Glu359, work as catalytic residues. The catalytic ability of these two residues is confirmed by biological assay of each mutant, E186Q and E359Q. The wild type and E186Q mutant structures complexed with various substrates show the substrate recognition mechanisms and the pyranose ring distortion at the subsite -1. These structural characters of Cel44A indicate that GH44 belongs to the clan GH-A, and that Cel44A's reaction is classified in retaining type hydrolysis.