title: Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III.
abstract: The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism.
authors.affiliation: Department of Applied Life Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan. mikami@kais.kyoto-u.ac.jp
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
authors.forename: Mizuho
authors.initials: M
authors.lastname: Ban
authors.forename: Sachiko
authors.initials: S
authors.lastname: Suzuki
authors.forename: Hye-Jin
authors.initials: HJ
authors.lastname: Yoon
authors.forename: Osamu
authors.initials: O
authors.lastname: Miyake
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamasaki
authors.forename: Kohei
authors.initials: K
authors.lastname: Ogura
authors.forename: Yukie
authors.initials: Y
authors.lastname: Maruyama
authors.forename: Wataru
authors.initials: W
authors.lastname: Hashimoto
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
mesh_terms: D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D055162:Biocatalysis; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020578:Sphingomonas / Q000201:enzymology*
chemical_list: D001051:Apoenzymes; D020035:Holoenzymes; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase