Mechanistic Studies on the Vitamin B 12 -Catalyzed Dechlorination of Chlorinated Alkenes

149

151

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ؎ 1 kDa, whereas the native molecular mass was 71 ؎ 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-␤-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 ؎ 0.1 mol of cobalamin, 0.6 ؎ 0.02 mol of cobalt, 7.1 ؎ 0.6 mol of iron, and 5.8 ؎ 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ؎ 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K m values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 ؎ 3.2, 23.7 ؎ 5.2, and 47 ؎ 10 M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.Tetrachloroethene (PCE), a frequently detected groundwater contaminant, is used by different bacteria as a terminal electron acceptor in a process called dehalorespiration (10,14,16,20,21,32,35,40,46). Six such isolates belong phylogenetically to the subphylum Firmicutes of the gram-positive bacteria, whereas other isolates are affiliated with phylogenetic groups such as the ␦ and ε subclasses of the Proteobacteria and the green no...

Section: Resultsmentioning

confidence: 89%

Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of Dehalobacterrestrictus

Maillard

Schumacher

Vazquez

et al. 2003

149

151

“…The kinetic isotope effect of hydrogen was much lower, suggesting a secondary isotope effect, which indicates that the protonation was not rate limiting during the overall reaction (10) and may occur subsequent to the dehalogenation. Identification of chloroethenylcobalamins as intermediates provides evidence for the formation of a covalent cobalt-carbon bond during dehalogenation (13,16,27,35,36). The chlorine pattern of the chloroethenylcobalamin (13) indicates that a chlorine has already been released, suggesting an irreversible reaction step.…”

Section: Discussionmentioning

confidence: 99%

“…The microbial dehalogenation by cobalamin-containing dehalogenases has been proposed to proceed by alkylating a superreduced corrinoid containing a Co(I) species at the reactive site with the chloroethene (26). The chemical mechanism of the reductive dehalogenation of chlorinated ethenes catalyzed by cobalamin has been the subject of previous studies and has been suggested to occur via a single electron transfer from a reduced cob(I)alamin, leading to the formation of chloride and vinyl radicals as intermediates (3,7,36,41). The cleavage of the carbon-chlorine bond may produce the primary carbon isotope effect during dehalogenation by cobalamin-containing dehalogenases.…”

mentioning

confidence: 99%

Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin

Nijenhuis¹,

Andert²,

Beck³

et al. 2005

127

167

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B 12 ) was studied. Fractionation was largest during the reaction with cyanocobalamin with ␣C ‫؍‬ 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (␣C ‫؍‬ 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (␣C ‫؍‬ 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, ␣C values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.The chlorinated ethenes tetrachloroethene (PCE) and trichloroethene (TCE) have been used as solvents in the drycleaning industry and as metal degreasing agents and are among the most common groundwater contaminants due to spillage and leakage (12). The assessment of in situ biodegradation of groundwater contaminants is difficult since a decrease in the concentration can be a result of many factors, including dilution, sorption, and biological conversion. Monitoring and assessment of in situ microbial degradation activities of contaminants at polluted sites is therefore a major challenge. In the last few years, the application of stable isotope techniques has been suggested for assessment of the in situ biodegradation of contaminants (for a review, see references 17 and 33).Stable isotope fractionation of PCE and TCE has been observed under field conditions in contaminated aquifers as well as in laboratory studies by mixed microbial cultures and microcosms (2, 9, 39, 40); however, the factors affecting the isotope fractionation have not yet been studied systematically.Several factors may influence the degree of stable isotope fractionation, including biodegrading microorganisms, properties of the dehalogenating enzymes, and the reaction mechanism. Cobalamins are important cofactors of reductive dehalogenases in organisms capable of dehalorespiration (8,10,15,20,25,26). The microbial dehalogenation by cobalamin-containing dehalogenases has been proposed to proceed by alkylating a superreduced corrinoid containing a Co(I) species at the reactive site with the chloroethene (26). The chemical mechanism of the reductive dehalogenation of chlorinated ethenes catalyzed by cobalamin has been the subject of previous studies and has been suggested to occur via a single elect...

“…Several RDases of organohalide-respiring strains belonging to the genera Desulfomonile, Dehalococcoides, Dehalobacter, Desulfitobacterium, and Sulfurospirillum were, at least partially, characterized (22,26,28,29). Except for the 3-chlorobenzoate RDase of Desulfomonile tiedjei strain DCB-1 (29), all characterized RDases of organohalide-respiring bacteria contain a cobalamin or cobamide (i.e., a cobalt-containing cyclic tetrapyrrole) cofactor, which is involved in electron transfer and the reduction of the chloro-organic electron acceptor (6,34). Dehalococcoides isolates grow and perform reductive dechlorination in completely synthetic, defined medium as long as cyanocobalamin (vitamin B 12 ) is provided (4).…”

mentioning

confidence: 99%

Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains

Yan

Ritalahti

Wagner

et al. 2012

119

120

ABSTRACTDehalococcoides mccartyistrains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems.Dehalococcoideslacks the ability forde novocorrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B12) for growth. In contrast,Geobacter lovleyi, which dechlorinates tetrachloroethene tocis-1,2-dichloroethene (cis-DCE), and the nondechlorinating speciesGeobacter sulfurreducenshave complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium.G. lovleyi-D. mccartyistrain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, andcis-DCE was dechlorinated to vinyl chloride and ethene concomitant withDehalococcoidesgrowth. In contrast, negligible increase inDehalococcoides16S rRNA gene copies and insignificant dechlorination occurred inG. sulfurreducens-D. mccartyistrain BAV1 or strain FL2 cocultures. Apparently,G. lovleyiproduces a cobamide that complementsDehalococcoides' nutritional requirements, whereasG. sulfurreducensdoes not. Interestingly,Dehalococcoidesdechlorination activity and growth could be restored inG. sulfurreducens-Dehalococcoidescocultures by adding 10 μM 5′,6′-dimethylbenzimidazole. Observations made with theG. sulfurreducens-Dehalococcoidescocultures suggest that the exchange of the lower ligand generated a cobalamin, which supportedDehalococcoidesactivity. These findings have implications forin situbioremediation and suggest that the corrinoid metabolism ofDehalococcoidesmust be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.