3 years ago

Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase

Heterologous Production and Purification of a Functional
Chloroform Reductive Dehalogenase
Helene Lebhar, Karl Fisher, Karl A. P. Payne, Christopher P. Marquis, Mike Manefield, Susanne Bohl, Matthew Lee, Bat-Erdene Jugder, David Leys
Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron–sulfur clusters and a corrinoid cofactor was demonstrated using UV–vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

Publisher URL: http://dx.doi.org/10.1021/acschembio.7b00846

DOI: 10.1021/acschembio.7b00846

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