Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

Abstract

f 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.

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

“…Additionally, HupL (DET0110) appeared in lower bands, suggesting that DET0110 and DET0112 do not comigrate. This finding is supported by a previous investigation that used blue native PAGE (BN-PAGE) gels to separate RDases from the D. mccartyi-containing KB-1 consortia in order to monitor reductive dechlorination activity (43). For the TCE-induced consortia in that study, proteomic sequencing of the active band identified homologs of DET0187 and DET0186 with the KB1 homolog of DET0112.…”

“…Blue native gel separations were performed by previously published methods (43). In brief, a stable pool of proteins was established by growing a 5-liter culture derived from the D2 mixed community at a continuous respiration rate of 2.5 M Cl Ϫ /h for 800 h. The culture was centrifuged and was frozen at Ϫ80°C.…”

“…Slices corresponding to dominant bands were excised from the two non-silvernitrate-developed lanes, stored in glacial acetic acid, and later destained of Coomassie blue. They were subsequently trypsin digested and were analyzed via liquid chromatography-tandem mass spectrometry at the Proteomics Facility at the Helmholtz Centre for Environmental Research, Leipzig, Germany, by following the methods and Mascot protein-scoring algorithms described by Tang et al (43).…”

Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases

“…Blue Native PAGE is a promising approach for examining in vitro reductive dehalogenase activity [76,81]. The current frontier of biochemical research on the reductive dehalogenases involves attempts to heterologously express reductive dehalogenase encoding genes and to engineer an OHRB with desirable substrate preferences.…”

Microbial Degradation of Some Halogenated Compounds: Biochemical and Molecular Features

“…The function of TCE RDase, TceA, in transforming TCE to ethene has also been identified [61]. VcrA and BvcA catalyze the transformation of DCE to ethene [62,63]. In addition to chloroethenes, RDases also could reduce other chlorinated compounds.…”