title: Mutational analysis of cutinase-like enzyme, Cut190, based on the 3D docking structure with model compounds of polyethylene terephthalate.
abstract: The cutinase-like enzyme, Cut190, from Saccharomonospora viridis AHK190 can degrade the inner block of polyethylene terephthalate (PET) in the presence of Ca2+, and its mutant, S226P/R228S, exhibited increased activity and higher thermostability. The crystal structures of the Cut190 S226P mutant in the absence and presence of Ca2+were determined, and revealed the large conformational change induced upon Ca2+binding. However, the substrate-bound 3D structures of Cut190 remained unknown. In this study, to determine the substrate-binding site and improve the enzyme activity, we first built 3D structures of a PET model compound bound to the crystal structures, using the distance restraints between the scissile carbonyl group of the compound and the catalytic site of the enzyme. We then mutated the putative substrate-binding site predicted from the models, and experimentally determined the enzymatic activities of the mutants for the model substrate poly(butylene succinate-co-adipate). The mutated sites with decreased activity were consistent with the putative binding sites predicted by the 3D model from the Ca2+-bound crystal structure, suggesting that the structure of the Ca2+-bound state represents the active state. Notably, we generated two mutants with significantly increased activities.
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawabata
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522, Japan. Electronic address: oda@kpu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
authors.affiliation: Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Kyoto 606-8585, Japan.
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D062105:Molecular Docking Simulation; D011093:Polyethylene Terephthalates / Q000378:metabolism*
chemical_list: D011093:Polyethylene Terephthalates; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
keywords: Enzyme activity; Modeling; Mutation; Polyethylene terephthalate; Substrate-binding site