Reaction Mechanism and Stereochemistry of γ-Hexachlorocyclohexane Dehydrochlorinase LinA

γ-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide γ-hexachlorocyclohexane (γ-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of γ-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from γ-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic γ-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic γ-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic γ-PCCH. δ-PCCH was detected as the only product of dehydrochlorination of δ-HCH. 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.