Comparative biochemistry of three recently evolved hexachlorocyclohexane dehydrochlorinase variants

Abstract

The synthetic compound hexachlorocyclohexane (HCH) was widely used as a pesticide for several decades from the 1940s onwards but concerns about detrimental off-target effects have led to progressive reductions in its use in most countries since the 1970s. Even so, its persistence in the environment has meant it remains a concern and bioremediation options are therefore being explored to rehabilitate HCH-contaminated environments. Part of the research towards bioremediation solutions has involved studies of the bacterial enzymes responsible for the biodegradation of HCH, one of the key enzymes being the HCH-dehydrochlorinase LinA. Previous work has suggested LinA can catalyse two successive eliminations of trans-diaxial H-Cl pairs from various HCH isomers. This thesis compares the biochemistry of three different types of LinA, namely the LinA-type1 LinA2B90A, the LinA-type2 LinA1B90A and the novel LinA-type3 LinALL02. LinALL02 was found to differ from LinA1B90A and LinA2B90A in several respects, but particularly in its preference to degrade the delta-HCH isomer over the gamma-HCH isomer in the first elimination reaction and its ability to produce all three trichlorobenzene isomers (TCBs) as eventual dead-end products following the second elimination reaction. Valines at position 44 and 64 of LinALL02 were hypothesised as having roles in these unique characteristics and this hypothesis was supported to a degree by empirical analysis of synthetic mutants at these positions. Additionally, differences were found between the three LinA enzymes in their degradation of the (+)-alpha-HCH versus (-)-alpha-HCH enantiomers, and greater preferences were observed in assays of whole E.coli cells expressing the enzymes than in assays with purified enzymes. The variants also differed in their enantiopreferences in the second elimination reactions, specifically among the beta-, gamma-, and delta-pentachlorocyclohexenes (PCCHs) which are respectively produced from alpha-, gamma-, and delta-HCH degradation in the first elimination reactions. While most of my data are consistent with the accepted view that LinA works by eliminating trans-diaxial H-Cl pairs, I found it necessary to invoke syn- and anti-1,4-elimination of H-Cl pairs to account for the activities seen with delta- and beta-PCCH, respectively. A possible role for LinA in the previously proposed spontaneous production of TCBs from the proximal product of PCCH degradation, tetrachlorocyclohexadiene (TCDN), was also discussed. Molecular dynamics trajectories were performed for the three enzymes with alpha-, gamma-, and delta-HCH, and their corresponding PCCHs, but it was found that the percentage of time which substrates spent in productive poses in the active sites of the enzymes were not correlated to the enzymes' activity on the substrates. Show more