Identification of Dehalobacter reductive dehalogenases that catalyse dechlorination of chloroform, 1,1,1-trichloroethane and 1,1-dichloroethane

Abstract

Two novel reductive dehalogenases (RDases) that are highly similar to each other but catalyse distinct dechlorination reactions were identified from Dehalobacter-containing mixed cultures. These two RDases were partially purified from crude protein extracts of anaerobic dechlorinating enrichment cultures using blue native polyacrylamide gel electrophoresis. Gel slices were assayed for dechlorinating activity, and associated proteins were identified using liquid chromatography tandem mass spectrometry with the metagenome of the parent culture as the reference database. The two RDases identified, annotated as CfrA and DcrA, share an amino acid identity of 95.2 per cent, but use different substrates: CfrA dechlorinates chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA), but not 1,1-dichloroethane; DcrA dechlorinates 1,1-dichloroethane, but not CF or 1,1,1-TCA. These two novel RDases share no more than 40 per cent amino acid identity to any other known or putative RDases, but both have a twin-arginine motif and two iron-sulfur binding motifs conserved in most RDases. Peptides specific to two putative membrane anchor proteins, annotated as CfrB and DcrB, were also detected in gel slices.

Figure 1.

Image of left two lanes of the BN-PAGE gel, showing molecular weight ladder and the first stained sample lane. Remaining identical sample lanes (unstained) from the same gel were sliced at specific positions consistently by aligning to the stained lane as indicated. Each gel slice was then used in dechlorination assays. BP, band position. (Online version in colour.)

Figure 2.

Results of BN-PAGE with protein samples from the CF subculture. (a) Quantification of dechlorination products in enzyme assays with gel slices. The arrow bars indicate the regular technical error in headspace GC measurements. Plus symbol indicates the positive control, where activity was measured using 20 µl of protein extracts equal to the volume loaded onto each BN-PAGE well. Black bars, DCM detected from CF; grey bars, 1,1-DCA detected from 1,1,1-TCA; white bars, CA detected from 1,1-DCA. (b) Counts of LC–MS/MS-detected peptide hits in gel slices when searched against curated RdhA and RdhB proteins derived from the ACT-3 metagenome. ‘N/A’ means the gel slice was not analysed.

Figure 3.

Results of BN-PAGE with protein samples from the DCA subculture. (a) Quantification of dechlorination products in enzyme assays with gel slices; legend as in figure 2. (b) Counts of LC–MS/MS peptide hits in gel slices when searched against curated RdhA and RdhB proteins derived from the ACT-3 metagenome. ‘N/A’ means the gel slice was not analysed.

Figure 4.

Results of BN-PAGE with protein samples from the ACT-3 culture. (a) Quantification of dechlorination products in enzyme assays with gel slices; legend as in figure 2. (b) Counts of LC–MS/MS peptide hits in gel slices when searched against curated RdhA and RdhB proteins derived from the ACT-3 metagenome. ‘N/A’ means the gel slice was not analysed.

Figure 5.

Amino acid sequence alignment for CfrA versus DcrA, and CfrB versus DcrB. Sequences shown are for CfrA and CfrB. Residues that differ in DcrA and DcrB are in bold on next line. The twin-arginine motif is boxed and the two iron–sulfur binding motifs are shaded. The dashed underline indicates the predicted signal peptide. The peptides detected by LC–MS/MS are underlined.

Figure 6.

Maximum-likelihood phylogenetic tree of the RDases that have functional characterization. The alignment was generated using the MUSCLE algorithm, and the tree generated using the PhyML plugin in Geneious under the WAG model of evolution. Bootstrap support values (from 100 bootstrap iterations) are indicated where greater than 50 per cent. The scale bar represents the average number of substitutions per site. For a complete tree of curated RDase sequences see the introductory chapter to this issue [35].