A bacterial chloroform reductive dehalogenase: purification and biochemical characterization

Abstract

SummaryWe report herein the purification of a chloroform (CF)‐reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23‐fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X‐100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS‐PAGE and MALDI‐TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein−1. The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV‐visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe‐4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

Subtle changes in the active site architecture untangled overlapping substrate ranges and mechanistic differences of two reductive dehalogenases

“…This observation is consistent with the inability of DcaA to convert VC or MCA as substrate and is also in line with earlier studies on growing cultures of D. dichloroeliminans DCA1 [26]. So far, an RDase-mediated conversion of 1,1,1-TCA was only reported for chloroform RDases, which are distantly related to DcaA and PceA [14,33,34]. 1).…”

“…The exchange of Arg357 to Gln displayed a rather negligible effect on both mechanisms (Fig. However, comparison of protein sequences revealed the replacement of the tyrosine by a cysteine or phenylalanine in RDases specialized on the halogen substitution at chloromethanes [14,33,34] (Fig. In addition, the positive effect on TBE conversion, as it was shown for the respective PceA mutant, was not observed in this case, indicating structural differences at the active sites of both enzymes leading to a different substrate positioning.…”

“…In contrast, the isomer 1,1,1-TCA was not utilized, suggesting that the enzyme is not able to perform b-eliminations, where only one halide substituent is removed as a result of proton abstraction. So far, an RDase-mediated conversion of 1,1,1-TCA was only reported for chloroform RDases, which are distantly related to DcaA and PceA [14,33,34]. In these cases, 1,1,1-TCA was converted to 1,1-DCA and subsequently MCA via halogen substitution.…”

“…Essential cofactors of RDases are a cobamide at the active site and two iron-sulfur clusters [4,5]. Thus, only a few representatives were characterized with regard to substrate spectrum and function [11][12][13][14][15]. The cytoplasmic precursor bears an N-terminal twin arginine translocation (Tat) signal peptide [9] for membrane transport.…”

“…Low growth yields of most organohalide-respiring bacteria and the presence of numerous RDase homologs in a single organism often hampered biochemical characterization of the enzymes [10,11]. Thus, only a few representatives were characterized with regard to substrate spectrum and function [11][12][13][14][15]. Recently, protocols for functional heterologous production of RDases or nonfunctional heterologous production and subsequent reconstitution of active enzyme have been reported [5,16,17].…”