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Title (Primary) Functional characterization of reductive dehalogenases using blue native polyacrylamide gel electrophoresis
Author Tang, S.; Chan, W.W.M.; Fletcher, K.E.; Seifert, J.; Liang, X.; Löffler, F.E.; Edwards, E.A.; Adrian, L.;
Journal Applied and Environmental Microbiology
Year 2013
Department ISOBIO; PROTEOM;
Volume 79
Issue 3
Language englisch;
POF III (all) F11;
UFZ wide themes ru3
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 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.
ID 13115
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=13115
Tang, S., Chan, W.W.M., Fletcher, K.E., Seifert, J., Liang, X., Löffler, F.E., Edwards, E.A., Adrian, L. (2013):
Functional characterization of reductive dehalogenases using blue native polyacrylamide gel electrophoresis
Appl. Environ. Microb. 79 (3), 974 - 981