Details zur Publikation |
Kategorie | Textpublikation |
Referenztyp | Zeitschriften |
DOI | 10.1128/AEM.01873-12 |
Titel (primär) | Functional characterization of reductive dehalogenases using blue native polyacrylamide gel electrophoresis |
Autor | Tang, S.; Chan, W.W.M.; Fletcher, K.E.; Seifert, J.; Liang, X.; Löffler, F.E.; Edwards, E.A.; Adrian, L. |
Quelle | Applied and Environmental Microbiology |
Erscheinungsjahr | 2013 |
Department | ISOBIO; PROTEOM |
Band/Volume | 79 |
Heft | 3 |
Seite von | 974 |
Seite bis | 981 |
Sprache | englisch |
UFZ Querschnittsthemen | 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. |
dauerhafte UFZ-Verlinkung | 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 10.1128/AEM.01873-12 |