Abstract

Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, ε-HBCD has the highest symmetry. This makes ε-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to ε-HBCD. LC-MS data indicate that all enzymes converted ε-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants kcat and enzyme specificities kcat/KM indicate that ε-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (−)β-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the ε-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention.

Highlights

  • Pure g-HCH was extracted from the crude product in later years while other HCHs remained as waste material (Vijgen et al, 2011)

  • Racemic mixtures of a, b- and g-HBCDs and the meso forms dand ε-HBCD were isolated from a technical HBCD mixture (Saytex HP-900 ®, mp 1⁄4 168e184 C) by normal phase liquid chromatography (LC) on silica with n-hexane/dichloromethane mixtures (Heeb et al, 2005)

  • More variations are observed for the halide binding sites (Fig. 1, blue), which in case of LinA1 consists of glutamine (Q20), tryptophan (W42) and leucine (L129) and in case of LinA2 of lysine (K20), tryptophan (W42) and arginine (R129)

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Summary

Introduction

Radical-induced halogenation of various hydrocarbon precursors has been exploited in the past to produce polyhalogenated compounds at large scales. This approach can produce molecular libraries of hundreds of compounds differing in constitution and stereochemistry. G-HCH (Lindane) is the active insecticide, present at about 8e15% in technical HCH mixtures (Li, 1999; Vijgen et al, 2011). In 2009, a-, b- and g-HCHs, the most abundant isomers in technical HCH mixtures, were included in the list of persistent organic pollutants (POPs) of the Stockholm Convention (SC) and HCH production and use were banned (UNEP, 2006; 2009)

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