Transformation of ε-HBCD with the Sphingobium Indicum enzymes LinA1, LinA2 and LinATM, a triple mutant of LinA2
| Autor: | Rup Lal, Norbert V. Heeb, Thomas Fleischmann, Jasmin Hubeli, Peter Lienemann, Hans-Peter E. Kohler, Namita Nayyar |
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| Rok vydání: | 2021 |
| Předmět: |
Environmental Engineering
Stereochemistry Health Toxicology and Mutagenesis 0208 environmental biotechnology 02 engineering and technology 010501 environmental sciences 01 natural sciences chemistry.chemical_compound Biotransformation Escherichia coli Environmental Chemistry Enzyme kinetics Flame Retardants 0105 earth and related environmental sciences chemistry.chemical_classification Public Health Environmental and Occupational Health Substrate (chemistry) Stereoisomerism General Medicine General Chemistry Pollution Hydrocarbons Brominated 020801 environmental engineering Amino acid Sphingomonadaceae Enzyme chemistry Enantiomer Lindane Hexachlorocyclohexane Sphingobium indicum |
| Zdroj: | Chemosphere. 267:129217 |
| ISSN: | 0045-6535 |
| Popis: | Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, e-HBCD has the highest symmetry. This makes e-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 e-HBCD. LC-MS data indicate that all enzymes converted e-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants kcat and enzyme specificities kcat/KM indicate that e-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 e-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. |
| Databáze: | OpenAIRE |
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