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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 k cat and enzyme specificities k cat / K M 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. Image 1 • Implementation of the LinA1 substrate-binding site to LinA2 by point-directed mutagenesis. • Expression of three LinA enzyme variants, LinA1, LinA2 and LinATM, in E. coli bacteria. • Three of eight HBCD stereoisomers are converted by the dehydrohalogenase LinA2. • All LinA variants follow Michaelis-Menten kinetics for the ε-HBCD transformation. • Stereoselective conversion of ε-HBCD to 1E,5S,6R,9S,10R-PBCDen by LinA2. [ABSTRACT FROM AUTHOR]

Short-chain chlorinated paraffins (SCCPs) are listed as persistent organic pollutants (POPs) under the Stockholm Convention. Such substances are toxic, bioaccumulating, transported over long distances and degrade slowly in the environment. Certain bacterial strains of the Sphingomonadacea family are able to degrade POPs, such as hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). The haloalkane dehalogenase LinB, expressed in certain Sphingomonadacea, is able to catalyze the transformation of haloalkanes to hydroxylated compounds. Therefore, LinB is a promising candidate for conversion of SCCPs. Hence, a mixture of chlorinated tridecanes was exposed in vitro to LinB, which was obtained through heterologous expression in Escherichia coli. Liquid chromatography mass spectrometry (LC-MS) was used to analyze chlorinated tridecanes and their transformation products. A chloride-enhanced soft ionization method, which favors the formation of chloride adducts [M+Cl]- without fragmentation, was applied. Mathematical deconvolution was used to distinguish interfering mass spectra of paraffinic, mono-olefinic and di-olefinic compounds. Several mono- and di-hydroxylated products including paraffinic, mono-olefinic and di-olefinic compounds were found after LinB exposure. Mono- (rt = 5.9–6.9 min) and di-hydroxylated (rt = 3.2–4.5 min) compounds were separated from starting material (rt = 7.7–8.5 min) by reversed phase LC. Chlorination degrees of chlorinated tridecanes increased during LinB-exposure from n Cl = 8.80 to 9.07, indicating a preferential transformation of lower chlorinated (Cl <9) tridecanes. Thus, LinB indeed catalyzed a dehalohydroxylation of chlorinated tridecanes, tridecenes and tridecadienes. The observed hydroxylated compounds are relevant CP transformation products whose environmental and toxicological effects should be further investigated. Image 1 • Chlorinated paraffins are transformed by the enzyme LinB. • Mono- and di-hydroxylated transformation products could be identified. • Mathematical deconvolution was used to obtain non-interfered data. • Mono- and di-hydroxylated products were separated from starting material by LC. • Preferential conversion of lower chlorinated CPs was induced by LinB. [ABSTRACT FROM AUTHOR]