title: Biochemical and structural analyses of a bacterialendo-β-1,2-glucanase reveal a new glycoside hydrolase family.
abstract: β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified anendo-β-1,2-glucanase to homogeneity from cell extracts of the environmental speciesChitinophaga arvensicola, and anendo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related speciesChitinophaga pinensisThe Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is anendo-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.
authors.affiliation: From the Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657.|the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.affiliation: the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, m-nakajima@rs.tus.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: the Faculty of Agriculture, Iwate University, Iwate 020-8550.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Matsunaga
authors.affiliation: the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.|the School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuoh-ku, Sagamihara 229-8501.
authors.forename: Shinji
authors.identifier: 0000-0001-8773-3909
authors.initials: S
authors.lastname: Kamisuki
authors.affiliation: the Faculty of Agriculture, Niigata University, Niigata 950-2181, and.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nihira
authors.affiliation: the Faculty of Agriculture, Niigata University, Niigata 950-2181, and.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: the Faculty of Agriculture, Niigata University, Niigata 950-2181, and.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Sugimoto
authors.affiliation: the Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan.
authors.forename: Akimasa
authors.identifier: 0000-0003-2219-6051
authors.initials: A
authors.lastname: Miyanaga
authors.affiliation: the Faculty of Agriculture, Niigata University, Niigata 950-2181, and.
authors.forename: Hiroyuki
authors.identifier: 0000-0002-4213-3130
authors.initials: H
authors.lastname: Nakai
authors.affiliation: From the Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657.
authors.forename: Takatoshi
authors.identifier: 0000-0001-6496-5440
authors.initials: T
authors.lastname: Arakawa
authors.affiliation: From the Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657.
authors.forename: Shinya
authors.identifier: 0000-0003-1346-6435
authors.initials: S
authors.lastname: Fushinobu
authors.affiliation: the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.
authors.forename: Hayao
authors.initials: H
authors.lastname: Taguchi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D041963:Bacteroidetes / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification; D018360:Crystallography, X-Ray
chemical_list: D001426:Bacterial Proteins; D002480:Cellulase
keywords: Chitinophaga pinensis; Gram-negative bacteria; endo-β-1,2-glucanase; enzyme structure; glycosidase; novel glycoside hydrolase family; oligosaccharide; polysaccharide; sophorooligosaccharide; β-1,2-glucan