Molecular Characterization of the PceA Reductive Dehalogenase of Desulfitobacterium sp. Strain Y51

Abstract

The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol · min ؊1 · mg of protein ؊1 . The apparent K m values for PCE and TCE were 105.7 and 535.3 M, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.Recently, a number of studies have described reductive dehalogenation by bacteria which occurs during the initial attack of halogenated hydrocarbons. Such bacteria can grow by anaerobic respiration, a process that has been referred to as halorespiration or dehalorespiration (6, 9, 11). Several dehalorespiring bacteria have been reported to catalyze the reductive dehalogenation of tetrachloroethene (also referred to perchloroethene [PCE]). Most of these bacteria reduce PCE or trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) (5,10,18,19). Dehalococcoides ethenogenes strain 195 is able to catalyze the reductive dehalogenation of PCE to ethene (16,17).PCE dehalogenases have been purified, and their genes were cloned from some anaerobic bacteria such as Dehalospirillum multivorans (21-23), Desulfitobacterium sp. strain PCE-S (20), Dehalobacter restrictus (26, 27), and D. ethenogenes 195 (13). The molecular characterization of the chloroethene reductive dehalogenases from phylogenetically distinct bacteria has revealed significant similarities in molecular masses (51 to 65 kDa) and functional domains, but it is also true that similarities of the entire amino acid sequences are surprisingly low among these enzymes. The PCE dehalogenase of Clostridium bifermentans DPH-1 was recently purified and sequenced (24). This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of DCE isomers along with PCE and TCE. All the PCE dehalogenase genes except pceC from C. bifermentans DPH-1 (24) characterized to date are preceded by twin arginine signal sequences (2,3,25). This class of proteins contains corrinoid and two Fe/S clusters as prosthetic groups (11, 31). The PCE dehalogenase genes were found to be linked with open reading frames (ORFs) coding for small hydrophobic proteins containing two or three transmembrane helices (14,23,28,30). It has been proposed that the PceB protein in D. multivorans might act as a membrane anchor that functionally links the dehalogenase to the respiratory c...

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…A second deep-branching group of RdhA sequences contains D. restrictus PceA (RdhA24) and two slightly more distant members (RdhA20 and -22). These proteins form a closely related family together with some of the best-characterized enzymes, namely PceA of several Desulfitobacterium hafniense isolates [4,73,74], DcaA of Desulfitobacterium dichloroeliminans [57] and CprA5 (dechlorinating 3,5-dichlorophenol) of Desulfitobacterium hafniense PCP-1 [75]. The last seven RdhA proteins build up a group of highly heterogeneous enzymes for which no characterized counterpart is yet available.…”

Combining Low-Energy Electrical Resistance Heating with Biotic and Abiotic Reactions for Treatment of Chlorinated Solvent DNAPL Source Area

“…This type of temperature function is well documented for microbial processes (Atlas and Bartha 1987;Empadinhas et al 2004;Suyama et al 2002), and for reductive dechlorination reactions in particular (Kohring et al 1989, Holliger 1993, He 2003, and was observed for ZVI dechlorination processes (section 5.3). Note that the rate of some reactions, such as hydrolysis, may also continue to increase with increasing temperature.…”

“…The added value of increasing temperatures may not only enhance dissolution further, but biological reaction rates increase with increasing temperature within a specific range of temperature. Microbial activity is a function of temperature, and for mesophilic microorganisms, which include Dehalococcoides ethenogenes (Empadinhas et al 2004) as well as other dehalogenators (Suyama et al 2002), optimal metabolic rates are typically near 30 to 40°C, which ERH can stimulate (Heath and Truex 1994). Figure 2- In addition, Figure 2-5 illustrates the response of DHC 16S rRNA gene and functional reductase genes tceA, bvcA, and vcrA at Fort Lewis NAPL Area 3 before and during ERH heating.…”

Microbial Degradation of Some Halogenated Compounds: Biochemical and Molecular Features

“…PceC contains six-transmembrane domains, an FMN binding domain, and a C-terminal polyferredoxin-like domain. It is similar to the membrane-binding transcription regulators [28,60]. More examples for the organization of different RDase gene clusters are presented in [28].…”

“…Reductive dechlorination reactions are catalyzed by reductive dehalogenases (RDases) The purified PCE RDase, PceA, has proved to transform PCE and TCE to cDCE [60]. The function of TCE RDase, TceA, in transforming TCE to ethene has also been identified [61].…”