Crystal Structure and Site-Directed Mutagenesis Analyses of

The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicumUT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of -hexachlorocyclohexane (-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclo-hexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98 % identity in amino acid se-quence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid resi-dues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from -HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how eachmutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases. Hexachlorocyclohexane (HCH) is a six-chlorine-substitutedcyclohexane. One of its isomers, the isomer, has insecticidal properties and has been widely used as an insecticide around the world (1). Although the use of -HCH has been prohibited in most countries due to its toxicity and long persistence, the large