Transformation of ε-HBCD with the Sphingobium Indicum enzymes LinA1, LinA2 and LinATM, a triple mutant of LinA2.

Autor: Heeb NV; Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Advanced Analytical Technologies, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland. Electronic address: norbert.heeb@empa.ch., Hubeli J; Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Advanced Analytical Technologies, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland; ZHAW, Zurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Reidbach, CH-8820, Wädenswil, Switzerland; Current Address: Cantonal Pharmacy Zürich, Südstrasse 3, CH-8952, Schlieren, Switzerland., Fleischmann T; Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, CH-8600, Dübendorf, Switzerland., Lienemann P; ZHAW, Zurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Reidbach, CH-8820, Wädenswil, Switzerland., Nayyar N; Sri Venkateswara College, University of Delhi, Delhi, 1110021, India., Lal R; The Energy and Resources Institute, India Habitat Center, New Delhi, Delhi, 110003, India., Kohler HE; Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, CH-8600, Dübendorf, Switzerland.
Jazyk: angličtina
Zdroj: Chemosphere [Chemosphere] 2021 Mar; Vol. 267, pp. 129217. Date of Electronic Publication: 2020 Dec 07.
DOI: 10.1016/j.chemosphere.2020.129217
Abstrakt: 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.
Competing Interests: Declaration of competing interest The authors do not have any conflicts of interest with other entities or researchers regarding a publication of their data. Any opinions, findings, conclusions and recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the funding agencies.
(Copyright © 2020 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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