Dichloromethane Dehalogenase Research Articles

Unveiling microbial community structure and metabolic pathway over carbon cloth-titanium nitride-polyaniline biocathode for effective dichloromethane transformation

Chlorinated volatile organic compounds (Cl–VOCs) have dramatically biotoxicity and environmental persistence due to the presence of chlorine atoms, seriously jeopardizing ecological security and human health. Dichloromethane (DCM) as a model pollutant, is widely applied in solvents, extractants and cleaning agents in the pharmaceutical, chemical and food industries. In this study, highly biocompatible and conductive carbon cloth-titanium nitride-polyaniline (CC–TiN-PANI) bioelectrodes were obtained for DCM degradation in microbial electrolysis cell (MEC). The good adhesion of TiN and PANI on the electrode surface was demonstrated. The degradation kinetics were fitted by the Haldane model, compared to the CC bioelectrode (0.8 h−1), the proportion of maximum degradation rates to half-saturation concentration (Vmax/Km) of CC-TiN (1.4 h−1) and CC-TiN-PANI (2.2 h−1) bioelectrodes were enhanced by 1.8 and 2.8 times, respectively. Microbial community structure analysis illuminated that the dominant genera on the biofilm were Alicycliphilus and Hyphomicrobium, and the abundance was enhanced significantly with the modification of TiN and PANI. The dechlorination of DCM to formaldehyde could be catalyzed by DCM dehalogenase (DcmA) or by haloalkane dehalogenase (DhlA). And further oxidized to formate: 1) direct catalyzed by formaldehyde dehydrogenase (FdhA); 2) conjugated with glutathione by S-(hydroxymethyl)-glutathione synthase (Gfa), S-(hydroxymethyl)-glutathione dehydrogenase (FrmA) and S-formyl-glutathione hydrolase (FrmB); 3) conjugation with tetrahydrofolate (H4F) and/or tetrahydromethanopterin.

Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics.

Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced.

Bioinformatics, bacterial expression and enzyme activity analyses of dichloromethane dehalogenase from Methylobacterium rhodesianum H13

Dechlorination is the key step in biological degradation of dichloromethane (DCM), which is catalyzed by the enzyme DCM dehalogenase. In this study, the detailed structure and function of DCM dehalogenase in Methylobacterium rhodesianum H13 were investigated. Our results indicate that DCM dehalogenase (C1505H2255N403O430S7) is a stable hydrophilic protein localized in the cytoplasm, with an isoelectric point of 6.35. They also indicate that DCM dehalogenase is a frizzled protein with 52.43% of amino acid residues exposed on its surface, and that alpha-helices are a main structural component (53.47%). As one of the theta class glutathione S-transferases (GSTs), DCM dehalogenase presents as a dimeric structure and shows strong substrate selectivity. The catalytic tunnel radius and length of DCM dehalogenase are both 1.4 Å. Glu42 and Val43 are two of the essential amino acids in both the catalytic tunnel and catalytic pockets. The amino acid sequence of DCM dehalogenase was predicted to be homologous among various microorganisms, and its active site was conserved with eight potential sites. The recombinant DCM dehalogenase was expressed in E. coli BL21 strain. The optimum conditions for DCM dehalogenase are pH 8, temperature of 30 °C and presence of Mn2+. All the obtained information of DCM dehalogenase will lay the foundation for further study of its structural transformation and functional improvement.

Molecular Docking and Site-Directed Mutagenesis of Dichloromethane Dehalogenase to Improve Enzyme Activity for Dichloromethane Degradation.

Dichloromethane (DCM) dehalogenase in bacterial cells can catalyze the degradation of deleterious DCM in environments. However, the utility of naturally occurring DCM dehalogenase is often limited due to low enzyme activity and content in living cells. In this study, the gene encoding DCM dehalogenase was cloned from Methylobacterium rhodesianum and overexpressed in Escherichia coli. Based on molecular docking analysis of DCM dehalogenase using DCM as the ligand, all of the target amino acid residues within substrate binding pocket and 10 conservative amino acid residues were individually mutated to Ala. After determination of activity, R120, L121, W128, and T146 were chosen for further saturation mutation. Results showed that dcmT146A, dcmT146R, and dcmT146Q have higher activities, whereas dcmL121A, dcmT146L, dcmL121Q, and dcmL121F have retained activities. Next, these seven mutants with a single mutation on amino acid residue were chosen for double mutation. It was found that the mutant of dcmL121A/T146R exhibits the highest activity increasing by 52.8% relative to wild type. Bioinformatic and experimental analyses revealed that the mutant variant dcmL121A/T146R bears the reduced steric hindrance in the active center with a decreased number of amino acid residues within binding pocket from 8 to 5 while overall hydrophilicity increased. In addition, the number of hydrophobic amino acid residues within substrate binding pocket increased while Km value decreased. It was speculated that all these changes in mutant variant dcmL121A/T146R may contribute to the increase in catalytic activity. It can be concluded that our goal-orientated manipulation through homology modeling, molecular docking, and site-directed mutagenesis is effective for improvement of DCM dehalogenase activity and investigation of correlation between structure and function.

Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae.

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar εCl of -5.2 ± 0.1‰, but a substantially lower εC of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual element C-Cl isotope correlations (ΛC/Cl = Δδ13C/Δδ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.

Cloning and characterization of dichloromethane dehalogenase from Methylobacterium rhodesianum for dichloromethane degradation

ABSTRACTThe gene encoding dichloromethane dehalogenase from Methylobacterium rhodesianum was cloned. Bioinformatic analysis showed that dichloromethane dehalogenase gene sequence from M. rhodesianum is almost identical to the one from Methylobacterium extorquens, with only one base difference. Dichloromethane dehalogenase was subsequently expressed in Escherichia coli BL21 (DE3) and purified. It was found that enzyme activity in recombinant cells was 3 times higher than that in the wild-type M. rhodesianum. Further investigation showed that recombinant dichloromethane dehalogenase was most active at 40°C at pH 7–8, and its KM was 10.96 mM when treated with dichloromethane as substrate. The fitted curve of dichloromethane degradation gave a Vmax of 0.43 mM/h of in 0.01 M phosphate buffer. Degradation efficiency of dichloromethane reached 86.11% within 20 h. In addition, it was found that degradation efficiency of dichloromethane was highly associated with glutathione concentration, supporting the reports that glutathione functions as coenzyme of dichloromethane dehalogenase for dichloromethane degradation.

Phylogeny Poorly Predicts the Utility of a Challenging Horizontally Transferred Gene in Methylobacterium Strains

Horizontal gene transfer plays a crucial role in microbial evolution. While much is known about the mechanisms that determine whether physical DNA can be transferred into a new host, the factors determining the utility of the transferred genes are less clear. We have explored this issue using dichloromethane consumption in Methylobacterium strains. Methylobacterium extorquens DM4 expresses a dichloromethane dehalogenase (DcmA) that has been acquired through horizontal gene transfer and allows the strain to grow on dichloromethane as the sole carbon and energy source. We transferred the dcmA gene into six Methylobacterium strains that include both close and distant evolutionary relatives. The transconjugants varied in their ability to grow on dichloromethane, but their fitness on dichloromethane did not correlate with the phylogeny of the parental strains or with any single tested physiological factor. This work highlights an important limiting factor in horizontal gene transfer, namely, the capacity of the recipient strain to accommodate the stress and metabolic disruption resulting from the acquisition of a new enzyme or pathway. Understanding these limitations may help to rationalize historical examples of horizontal transfer and aid deliberate genetic transfers in biotechnology for metabolic engineering.

Analysis of the 3′-region of the dcmA gene of dichloromethane dehalogenase of Methylobacterium dichloromethanicum DM4

Two hypothetical genes of a dichloromethane (DCM) degrader Methylobacterium dichloromethanicum DM4, METDI2657 and METDI2658, located in the 3′-region of the dcmA gene of DCM dehalogenase, have been studied. The method of reverse transcription polymerase chain reaction (RT-PCR) was used to show that the cells of M. dichloromethanicum DM4 grown on both DCM and methanol contained the transcripts of all three of the above genes. RT-PCR amplification of the intergenic regions showed that the genes dcmA, METDI2657, and METDI2658 formed an operon. Orthologs of the METDI2657 and METDI2658 genes were also found in the DCM destructors Methylopila helvetica DM9, Methylorhabdus multivorans DM13 and DM15, Ancylobacter dichloromethanicus DM16, and Methylobacterium extorquens DM17. A mobilized suicide vector pK18mob was used to obtain a knock-out mutant of M. dichloromethanicum DM4 (NOK353) with the “turned-off” METDI2657 gene, the nucleotide sequence of which was interrupted by insertion of a gentamycin cassette. After cultivation on methanol, the NOK353 mutant had a lower rate of growth on DCM than the wild type strain DM4.

Chloride‐associated adaptive response in aerobic methylotrophic dichloromethane‐utilising bacteria

Aerobic methylotrophic bacteria able to grow with dichloromethane (DCM) as the sole carbon and energy source possess a specific glutathione S-transferase, DCM dehalogenase, which transforms DCM to formaldehyde, used for biomass and energy production, and hydrochloric acid, which is excreted. Evidence is presented for chloride-specific responses for three DCM-degrading bacteria, Methylobacterium extorquens DM4, Methylopila helvetica DM6 and Albibacter methylovorans DM10. Chloride release into the medium was inhibited by sodium azide and m -chlorophenylhydrazone, suggesting an energy-dependent process. In contrast, only nigericin affected chloride excretion in Mb. extorquens DM4 and Mp. helvetica DM6, while valinomycin had the same effect in A. methylovorans DM10 only. Chloride ions stimulated DCM-dependent induction of DCM dehalogenase expression for Mp. helvetica DM6 and A. methylovorans DM10, and shortened the time for onset of chloride release into the medium. Striking chloride-containing structures were observed by electron microscopy and X-ray microanalysis on the cell surface of Mp. helvetica DM6 and A. methylovorans DM10 during growth with DCM, and with methanol in medium supplemented with sodium chloride. Taken together, these data suggest the existence of both general and specific chloride-associated adaptations in aerobic DCM-degrading bacteria.

Identification and cloning of a gene encoding dichloromethane dehalogenase from a methylotrophic bacterium, Bacillus circulans WZ-12 CCTCC M 207006

The gene dehalA encoding a novel dichloromethane dehalogenases (DehalA), has been cloned from Bacillus circulans WZ-12 CCTCC M 207006. The open reading frame of dehalA, spanning 864 bp, encoded a 288-amino acid protein that showed 85.76% identity to the dichloromethane dehalogenases of Hyphomicrobium sp. GJ21 with several commonly conserved sequences. These sequences could not be found in putative dichloromethane (DCM) dehalogenases reported from other bacteria and fungi. DehalA was expressed in Escherichia coli BL21 (DE3) from a pET28b(+) expression system and purified. The subunit molecular mass of the recombinant DehalA as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 33 kDa. Subsequent enzymatic characterization revealed that DehalA was most active in a acidic pH range at 30 degrees , which was quite different from that observed from a facultative bacterium dichloromethane dehalogenases of Methylophilus sp. strain DM11. The Michaelis-Menten constant of DCM dehalogenase was markedly lower than that of standard DCM dehalogenases.

Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane

Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.

Bacillus circulans WZ-12—a newly discovered aerobic dichloromethane-degrading methylotrophic bacterium

A novel dichloromethane (DCM)-degrading bacterial strain named WZ-12 (GenBank accession no. EF100968) was isolated and identified as Bacillus circulans based on standard morphological and physiological properties and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. DCM dehalogenase from B. circulans WZ-12 was purified to 8.27-fold with a yield of 34.83%. The electrophoretically homogeneous-purified enzyme exhibited a specific activity of 118.82 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified DCM dehalogenase gave a distinct band with an estimated molecular mass of 20,000 +/- 1,000.

Adaptation of aerobic methylobacteria to dichloromethane degradation

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.

Effects of DNA-damaging Agents on Aerobic Methylobacteria Capable and Incapable of Utilizing Dichloromethane

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

Molecular characterization of dichloromethane-degrading Hyphomicrobium strains using 16S rDNA and DCM dehalogenase gene sequences

A phylogenetic analysis of 6 strains of dichloromethane (DCM) utilizing bacteria was performed. Based on the almost complete 16S rDNA sequence determination, all strains clustered together and showed high sequence similarity to Hyphomicrobium denitrificans, except for the strain MC8b, which is only moderately related to them and probably represents a distinct species. The 16S rDNA-based phylogenetic tree was compared to the one obtained from the DNA sequence data of the dcmA gene coding DCM dehalogenase, the key enzyme of DCM utilization. The topology of the two trees is in good agreement and may suggest an ancient origin of DCM dehalogenase, but also raises questions about the original role of the enzyme.

Physiological and Biochemical Analysis of the Transformants of Aerobic Methylobacteria Expressing the dcmA Gene of Dichloromethane Dehalogenase

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.

Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11.

The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp. strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl). The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde. With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)). The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1). The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step. The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt. The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well. The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water). Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species. Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.

Dichloromethane metabolism and C1 utilization genes in Methylobacterium strains.

The ability of methylotrophic alpha-proteobacteria to grow with dichloromethane (DCM) as source of carbon and energy has long been thought to depend solely on a single cytoplasmic enzyme, DCM dehalogenase, which converts DCM to formaldehyde, a central intermediate of methylotrophic growth. The gene dcmA encoding DCM dehalogenase of Methylobacterium dichloromethanicum DM4 was expressed from a plasmid in closely related Methylobacterium strains lacking this enzyme. The ability to grow with DCM could be conferred upon Methylobacterium chloromethanicum CM4, a chloromethane degrader, but not upon Methylobacterium extorquens AM1. In addition, growth of strain AM1 with methanol was impaired in the presence of DCM. The possibility that single-carbon (C1) utilization pathways in dehalogenating Methylobacterium strains differed from those discovered in strain AM1 was addressed. Homologues of tetrahydrofolate-linked and tetrahydromethanopterin-linked C1 utilization genes of strain AM1 were detected in both strain DM4 and strain CM4, and cloning and sequencing of several of these genes from strain DM4 revealed very high sequence identity (96.5-99.7%) to the corresponding genes of strain AM1. The expression of transcriptional xylE fusions of selected genes of the tetrahydrofolate- and tetrahydromethanopterin-linked pathways from strain DM4 was investigated. The data obtained suggest that the expression levels of some C1 utilization genes in M. dichloromethanicum DM4 grown with DCM may differ from those observed during growth with methanol.

The elusive roles of bacterial glutathione S-transferases: new lessons from genomes.

Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals. Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates. In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals. Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds. Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria. In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes. Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated.

Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: mono- and dihalomethanes.

A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation. Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO. Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases. Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH(3)Cl and using in vitro assays (HCHO formation) and the S. typhimurium mutagenesis assay with the dihalomethanes CH(2)Cl(2), CH(2)Br(2), CH(2)BrCl, CH(2)ICl, CH(2)I(2), and CH(2)ClF. GSTs 5-5 and T1 had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested. In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested. A similar pattern was observed for the conjugation of CH(3)Cl. The reversion tests with S. typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5. Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic. The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue [Wheeler, J. B., Stourman, N. V., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1107-1117]. The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps.