Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers

Abstract

LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme ‘hexachlorocyclohexane (HCH)-dehydrochlorinase’. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10532-017-9786-9) contains supplementary material, which is available to authorized users.

Seeking the Source of Catalytic Efficiency of Lindane Dehydrochlorinase, LinA

“…Interestingly, significant differences between type I and type II complexes were observed in terms of HCH isomers interactions with residues reported as relevant for enantioselectivity (K20Q, L96C, A131G, and T133M). 25 Our results indicate that interaction with residue 20 is among the strongest ones in every type I complex while much smaller in type II complexes. Similarly, L96 contributes to favorable enzyme–substrate interactions in all type I complexes, unlike C96 in LinA-type II that does not interact with γ- and (+)-α- isomers and causes unfavorable interaction with the (−)-α- isomer.…”

“…Importantly, a less significant effect was observed in the case of (+)-α-HCH complex which points to differences in the interaction pattern with the protein between this substrate and the two others, more efficiently metabolized by LinA-type I. 24 , 25 , 27 …”

“…On the basis of the subsequent mutagenesis studies, it has been suggested that 4 out of these 10 residues vicinal to the active site of LinA might govern the enantioselectivity toward α-HCH enantiomer but do not necessarily play a similar role in the transformation of γ-HCH. 25 …”

“…However, the complete change of enantioselectivity has not been reached yet, so the molecular factors responsible for this behavior have not been fully described and understood. 25 All those results have contributed to disclosing the molecular mechanism of LinA enzymes and making a step forward toward improving their efficiency; however there are still many key aspects unaddressed. As long as the source of the differences between LinA variants remains elusive, any rational modification of the enzyme in order to improve it toward selected isomers will be prohibited.…”

Genomic Analysis of γ-Hexachlorocyclohexane-Degrading Sphingopyxis lindanitolerans WS5A3p Strain in the Context of the Pangenome of Sphingopyxis

“…Other LinA type-1a residues are the same as in LinA type-1. Based on the presence of K20, L96, and A131 residues we can predict that LinA type-1a will exhibit preference to the (−) enantiomers [76].…”