title: Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.
abstract: The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Okai
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.forename: Lica Fabiana
authors.initials: LF
authors.lastname: Imai
authors.forename: Tomoko
authors.initials: T
authors.lastname: Mase
authors.forename: Ryota
authors.initials: R
authors.lastname: Moriuchi
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Koji
authors.initials: K
authors.lastname: Nagata
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003511:Cyclohexanols / Q000378:metabolism; D001556:Hexachlorocyclohexane / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D042301:Sphingomonadaceae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C525117:2,3,4,5,6-pentachlorocyclohexanol; C525116:2,3,5,6-tetrachlorocyclohexane-1,4-diol; D003511:Cyclohexanols; D001556:Hexachlorocyclohexane; D006867:Hydrolases; C046137:haloalkane dehalogenase; C023888:beta-hexachlorocyclohexane
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