S8: Ascribing function to reductive dehalogenase genes

Organohalide-respiring bacteria such as Dehalococcoides, Dehalobacter and Dehalogenimonas are strict anaerobes harboring multiple distinct putative reductive dehalogenase genes within their genomes. While hundreds of putative reductive dehalogenase gene sequences have been identified, only a handful have been functionally characterized because of difficulties inherent to working with slow-growing strict anaerobes, the lack of genetic systems to manipulate these organisms, and the inability to express functional reductive dehalogenases heterologously.  We have been using blue native polyacrylamide gel electrophoresis (BN-PAGE) to separate proteins from cell-free extracts followed by reductive dehalogenation enzyme assays to functionally characterize the substrates for specific reductive dehalogenases.  Finally, liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to identify proteins.  Using this approach, we have extended the substrate ranges for known Dehalococcoides chlorinated ethene dehalogenases.  In addition, we have identified novel Dehalobacter and Dehalocccoides reductive dehalogenases. Two novel Dehalobacter enzymes, annotated as CfrA and DcrA, share 95.2% amino acid sequence identity, but do not share substrates. CfrA dechlorinates chloroform and 1,1,1-trichloroethane, but not 1,1-dichloroethane, while DcrA dechlorinates 1,1-dichloroethane, but not chloroform or 1,1,1-trichloroethane. The dcpA gene identified in several Dehalocccoides strains encodes DcpA, a reductive dehalogenase that converts 1,2-dichloropropane to propene. The BN-PAGE approach combined with LC-MS/MS analysis provides a path forward to begin to explore this largely unknown protein family.