title: Functional characterization of reductive dehalogenases by using blue native polyacrylamide gel electrophoresis.
abstract: Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Shuiquan
authors.initials: S
authors.lastname: Tang
authors.forename: Winnie W M
authors.initials: WW
authors.lastname: Chan
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Fletcher
authors.forename: Jana
authors.initials: J
authors.lastname: Seifert
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Liang
authors.forename: Frank E
authors.initials: FE
authors.lastname: Löffler
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Edwards
authors.forename: Lorenz
authors.initials: L
authors.lastname: Adrian
mesh_terms: D041862:Chloroflexi / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D002853:Chromatography, Liquid; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006219:Halogens / Q000378:metabolism*; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D063347:Native Polyacrylamide Gel Electrophoresis; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D006219:Halogens; D006843:Hydrocarbons, Chlorinated; D006867:Hydrolases