Characterization of hexachlorocyclohexane metabolizing lin genes from a soil metagenome

Abstract

The present study was aimed at evaluation of linA and linB diversity in the metagenome of a HCH-contaminated soil, and their characterization by PCR amplification approach. Results have revealed that indeed several linA variants were present in the soil metagenome, many of which were novel from previously reported ones. Relative abundance of two genes, named as linA-type1 and linA-type2, was highest (10 and 18% respectively) Primary sequence of linA-type1 is 100% identical to reported linA-UT26. Gene linA-type2 is novel and its sequence is identical to the reported linA1-B90 in 1-443 bp region, except for one change C376T, but to linA-UT26 in downstream region. High abundance of linA-type1and -type2, and presence of several variants where the nucleotide changes were not random but restricted to selected locations that are present between linand#913;-type1 and -type2, suggests that either linA-type1or -type2 variants could have converted into one another by accumulation of adaptive mutations, or the chimeric variants were formed by recombination between linA-type1and -type2 at different locations. The result also suggests that presence of different Cterminal region of LinA1-B90 has no role in this property. This enantioselectivity is retained by shorter versions i.e. LinA-type1and#916; and -type2and#916;, suggesting that the repeated motif has no role in this property. LinA-type2 was found to be substantially more thermostable than LinA-type1, possibly due to the presence of an additional salt bridge R31-E81and#8242; that was identified in its crystal structure. Overall, the study unveiled numerous variants of linA and linB, many of whom were previously not described. Encoded products of some of these have distinct properties of thermostability or enantioselectivity. Further, it paves way for designing better enzymes for improved degradation of HCH-isomers.