| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
γ-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level, and for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of enzymes containing scytalone dehydratase, 3-oxo-Δ5-steroid isomerase, nuclear transport factor-2, and the β-subunit of naphthalene dioxygenase-all known structures with different functions. The three-dimensional structure of LinA is predicted by homology modeling. More than twenty mutants were prepared by site-directed mutagenesis. These mutants were expressed in E.coli, and their activities in crude extract were evaluated. Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity. We conclude that LinA catalyses the proton abstraction via the catalytic dyad H73-D25 by the similar mechanism as described for scytalone dehydratase. The results suggest that LinA and scytalone dehydratase evolved from a common ancestor. LinA may have evolved from an enzyme showing a dehydratase activity.
Stereochemical analysis of the reaction products formed during conversion of γ-HCH by LinA was investigated by GC-MS, NMR, CD and molecular modeling. LinA requires the presence of a 1, 2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1, 2-biaxial HCl pairs present on γ-HCH and gives rise to a single PCCH enantiomer 1, 3 (R), 4 (S), 5(S), 6 (R)-PCCH.Furthermore, LinA enantiomerically differentiates 1, 3 (S), 4 (R), 5 (R), 6 (S)-PCCH and 1, 3 (R), 4 (S), 5 (S), 6 (R)-PCCH.The proposed mechanism of enzymatic biotransformation of γ-HCH to 1, 2, 4-TCB by LinA consists of two 1, 2-anti conformationally dependent dehydrochlorinations followed by 1, 4-anti dehydrochlorination.
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