Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Abstract

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K m value for PCP was 46.7 M at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.Halogenated compounds are generally known as toxic environmental pollutants. Hydrogenolytic reductive dehalogenation, a reaction involving the replacement of one halogen atom with one hydrogen atom, is the predominant mechanism for their transformation in anaerobic environments. This process can sustain microbial growth via electron transport-coupled phosphorylation (10,26,31). The majority of the known reductive dehalogenases (RDases) belong to the CprA/PceA family. These are single-polypeptide membrane-associated anaerobic enzymes that are synthesized as preproteins with a cleavable twin arginine translocation (TAT) peptide signal. They contain one corrinoid and two iron-sulfur clusters as cofactors.CprA enzymes catalyzing the reductive dechlorination of chloroaromatics have been purified from Desulfitobacterium hafniense strain DCB-2 (6), Desulfitobacterium dehalogenans (30), Desulfitobacterium chlororespirans strain Co23 (12, 14), Desulfitobacterium sp. strain PCE1 (29) (20) and characterized. However, none of these enzymes showed high dechlorinating activity toward highly chlorinated phenols such as pentachlorophenol (PCP).D. hafniense strain PCP-1 is the only known strict anaerobic bacterium which reductively dechlorinates PCP to 3-chlorophenol (3-CP) and a variety of halogenated aromatic compounds at the ortho,...

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

“…These RDases are distributed across the RdhA tree, and are known to catalyse the dehalogenation of a broad range of substrates, including chlorinated ethenes (VcrA, BvcA, TceA, MbrA, PceA, PrdA [38,40,54,60,74,93,100]), chlorinated ethanes (CfrA, DcrA, DcaA [73,81,94]), and chlorinated aromatics (CbrA, CprA [31,71,75,101]). For most characterized reductive dehalogenases, the full substrate range is not known; instead activity on a specific substrate has been verified.…”

“…See electronic supplementary material, figure S1 for a rectangular version of this phylogeny with scale bar and bootstrap support values for this tree. Reductive dehalogenases with known function on specific substrates, based on protein isolation, proteomics or transcriptomics are numbered as follows: (1) variety of chlorinated phenols [31,37,39,71]. Substrate specificities towards ortho or meta and para dechlorination are observed for the different CprAs.…”

Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill

“…Sequence alignment indicated that the assigned protein is the reductive dehalogenase RdhA3 of D. hafniense strain DCB‐2 T (B8FVX4) or the almost identical CprA5 of D. hafniense strain PCP‐1 (Q6V7J3, Table 3). Both are able to dehalogenate 2,4,5‐trichlorophenol (Bisaillon et al ., 2010; Mac Nelly et al ., 2014). Without yeast extract, this protein group was unique to the culture with 2,4,5‐T ( P  = 0.02).…”

“…Only one additional reductive dehalogenase was detected in the metaproteome data, which showed high similarity to a putative reductive dehalogenase (Q8RPG3, D. hafniense strain DCB‐2 T ) and to the pentachlorophenol reductive dehalogenase CprA3 (Q9ANS1, D. hafniense strain PCP‐1). CprA3 is not able to dechlorinate 2,4,5‐TCP (Bisaillon et al ., 2010). Still, this protein group was only detected when cells were cultivated with 2,4,5‐T, irrespective of whether yeast extract was present in the medium ( P  > 0.05), but its abundance was below the median of all protein groups and approximately 10‐fold lower than RdhA3/CprA5.…”

“…The cprA3 gene was shown to be transcriptionally induced by 2,4,6‐trichlorophenol (Bisaillon et al ., 2011) but was also transcribed in the absence of chlorophenols (Gauthier et al ., 2006). Interestingly, the purified CprA3 of strain PCP‐1 dechlorinated highly chlorinated phenols such as penta‐ and tetrachlorophenols and some trichlorophenols, but not 2,4,5‐TCP (Bisaillon et al ., 2010). This, together with the low amount of the corresponding dehalogenase in the metaproteome data, suggests an unspecific synthesis of this dehalogenase.…”