Reaction mechanism and stereochemistry of γ-hexachlorocyclohexane dehydrochlorinase LinA

gamma -Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma -hexachlorocyclohexane (gamma -HCH) by the soil bacterium Sphingomonas paucimobilis UT26, Stereochemical analysis of the reaction products formed during conversion of gamma -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 gamma -HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma -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 gamma -PCCH, 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma -PCCH, delta -PCCH was detected as the only product of dehydrochlorination of delta -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 gamma -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 gamma -HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.