title: Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding.
abstract: To identify the amino acids responsible for the substrate binding of chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), Tyr148 and Lys218 of the chitosanase were mutated to serine and proline, respectively, and the mutated chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.16% of the wild type, respectively. When the (GlcN)3 binding ability to the chitosanase was evaluated by fluorescence spectroscopy and thermal unfolding experiments, the binding abilities of both mutant enzymes were markedly reduced as compared with the wild type enzyme. The affinity of the enzyme for the trisaccharide decreased by 1.0 kcal/mol of binding free energy for Y148S, and 3.7 kcal/mol for K218P. The crystal structure of K218P revealed that Pro218 forms a cis-peptide bond and that the state of the flexible loop containing the 218th residue is considerably affected by the mutation. Thus, we conclude that the flexible loop containing Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less critical, but still important, due to a stacking interaction or hydrogen bond.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505. fukamizo@nara.kindai.ac.jp
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.forename: Satoko
authors.initials: S
authors.lastname: Amano
authors.forename: Kei
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Tomoye
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Tomomi
authors.initials: T
authors.lastname: Katsumi
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Saito
authors.forename: Michihiko
authors.initials: M
authors.lastname: Suzuki
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
authors.forename: Yoshiho
authors.initials: Y
authors.lastname: Nagata
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D006026:Glycoside Hydrolases; C019868:chitosanase