Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from Desulfitobacterium frappieri PCP-1

Abstract

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K m for 3,5-dicholorophenol was 49.3 ؎ 3.1 M at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.Several strictly anaerobic bacteria are able to reductively dehalogenate a large variety of chlorinated compounds and use them as terminal electron acceptors (8). Desulfomonile tiedjei DCB-1, Dehalobacter restrictus PER-K23, Sulfurospirillum (formerly Dehalospirillum) multivorans, and many members of the genus Desulfitobacterium have been the most studied for their dechlorinating activity. Three types of reductive dehalogenases have been isolated from dehalorespiring bacteria. The most frequently reported dehalogenases consist of a single polypeptide containing one corrinoid cofactor and two iron-sulfur clusters: tetrachloroethene (PCE) reductive dehalogenases of S. multivorans (18), Desulfitobacterium sp. strain PCE-S (16), and Desulfitobacterium frappieri TCE-1 (27), ortho-chlorophenol reductive dehalogenases of Desulfitobacterium hafniense (5), Desulfitobacterium dehalogenans (28), and Desulfitobacterium chlororespirans (10), and PCE-and trichloroethene (TCE)-reductive dehalogenases of Dehalococcoides ethenogenes (14). Two reductive dehalogenases with one corrinoid cofactor but without an iron-sulfur cluster have also been reported: the ortho-chlorophenol reductive dehalogenase from D. frappieri PCP-1 (3) and the PCE reductive dehalogenase from Clostridium bifermentans DPH-1 (22). These two proteins are d...

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

“…strain PCE-1 [72]. Another unique chlorophenol reductive dehalogenase is CprA5 found in Desulfitobacterium hafniense strains PCP-1, TCP-A and DCB-2, which has dechlorination activity against several chlorophenols at all three substituent (ortho, meta and para) positions [39,72]. A 1,2-dichloroethane (1,2-DCA)-specific reductive dehalogenase was identified in Desulfitobacterium dichloroeliminans strain DCA1 and also in the metagenome of a 1,2-DCA organohalide-respiring enrichment culture from a contaminated aquifer [73].…”

“…Several molecular tools have been developed for examining a broad range of rdhA genes in a given sample (site, isolate or mixed culture), including both microarray-based methods and PCR-based protocols [60][61][62][63]. Identification of novel reductive dehalogenase genes has primarily come from PCR studies [32,33,[35][36][37]39,40,42] and subsequently from genome and metagenome sequencing efforts [6,13,14,41,63], with each new organism or environment sampled increasing the known diversity of putative rdhA genes.…”

“…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.…”

“…The first gene sequences encoding reductive dehalogenases were identified using classical reverse genetics approaches based on partial amino acid sequences of purified enzymes [30,31]. PCR and genome studies have since identified new sequences with high sequence identity to the characterized genes [6,13,14,[32][33][34][35][36][37][38][39][40][41][42]. Reductive dehalogenase genes (or reductive dehalogenase homologous genes (rdh) if the encoded protein has not yet been biochemically characterized) typically comprise an operon containing rdhA, the gene for the catalytically active enzyme (RDase if characterized, otherwise RdhA), rdhB, a gene encoding a putative membrane-anchoring protein [34], and sometimes one or more members of rdhTKZECD, genes associated with reductive dehalogenase genes [43].…”

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

“…Both enzymes dehalogenated 3,5‐DCP at the meta ‐position and exhibited highest activity with this compound but they also accepted 2,4,5‐TCP as substrate. The dehalogenating activity of RdhA3 and CprA5 on 2,4,5‐TCP was one‐third and one half of the activity on 3,5‐dichlorophenol respectively (Thibodeau et al ., 2004; Mac Nelly et al ., 2014). In these studies, 2,4,5‐TCP was dechlorinated at the ortho position leading to 3,4‐DCP as the final product, as was observed in our enrichment cultures.…”