Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceA_TCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.     

The organohalide-respiring bacterium <Emphasis Type="Italic">Sulfurospirillum multivorans</Emphasis>: a natural source for unusual cobamides

Cobamides (‘complete’ corrinoids) are essential for organohalide-respiring bacteria because they act as cofactors of reductive dehalogenases (RDases). RDases are the key enzymes in organohalide respiration, a process relevant for environmental remediation. More than a decade ago, the unusual norpseudo-B₁₂ was identified as cofactor of the tetrachloroethene RDase (PceA) purified from the epsilonproteobacterium Sulfurospirillum multivorans. Since then, the question was raised whether or not the production of the uncommon cobamide is a specific adaptation to the requirements of PceA. Recently, efforts were made to unravel variations in the cobamide biosynthetic pathway, which lead to the production of the structurally unique norpseudo-B₁₂. The acquisition of genomic and proteomic data together with structural analyses of PceA provided insights into norcobamide formation and utilization. By the use of guided biosynthesis, S. multivorans was shown to be an effective cobamide producer capable of generating unusual norcobamides either functional or non-functional as cofactors of PceA. The organism turned out to be a suitable tool for testing the impact of cobamide structure on enzyme function. The results summarized here highlight S. multivorans in particular and the organohalide-respiring bacteria in general as a resource for new discoveries on cobamide diversity and utilization.     

Comparative proteomics of Dehalococcoides spp. reveals strain-specific peptides associated with activity

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.     

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

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-beta-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 micro 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.     

Reductive dechlorination of tetrachloroethene to ethene by a two-component enzyme pathway

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.     

Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.     

Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51

Desulfitobacterium hafniense strain Y51 dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by the action of the PceA reductive dehalogenase encoded by pceA. The pceA gene constitutes a gene cluster with pceB, pceC, and pceT. However, the gene components, except for pceA, still remained to be characterized. In the present study, we characterized the function of PceT. PceT of strain Y51 showed a sequence homology with trigger factor proteins, although it is evolutionally distant from the well-characterized trigger factor protein of Escherichia coli. The PceT protein tagged with 6x histidine was expressed as a soluble form in E. coli. The recombinant PceT fusion protein exhibited peptidyl-proryl cis–trans isomerase activity toward the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The PceT fusion protein also exhibited chaperon activity towards the chemically denatured citrate synthase. Immunoprecipitation analysis using antibodies raised against PceA and PceT demonstrated that PceT specifically binds to the precursor form of PceA with an N-terminal twin-arginine translocation (TAT) signal sequence. On the other hand, PceT failed to bind the mature form of PceA that lost the TAT signal sequence. This is the first report in dehalorespiring bacteria, indicating that PceT is responsible for the correct folding of the precursor PceA.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 microM and as low as 0.06 microM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min(-1) mg protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Reductive Dechlorination of Tetrachloroethene to Ethene by a Two-Component Enzyme Pathway

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium(III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 μmol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 μmol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.     

Development of primers for amplifying genes encoding CprA- and PceA-like reductive dehalogenases in anaerobic microbial consortia, dechlorinating trichlorobenzene and 1,2-dichloropropane

Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I-IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.     

Molecular characterization of the PceA reductive dehalogenase of desulfitobacterium sp. strain Y51

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 x min(-1) x mg of protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, 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.     

A novel method for expression and large-scale production of human brain l-glutamate decarboxylase

l-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases. Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme. Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67. This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms. This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems.     

Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin-dependent transport-coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO₂ reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H₂ consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.     

Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.