Aerobic and Anaerobic Biodegradation of 1,2-Dibromoethane by a Microbial Consortium under Simulated Groundwater Conditions.

This study was conducted to explore the potential for 1,2-Dibromoethane (EDB) biodegradation by an acclimated microbial consortium under simulated dynamic groundwater conditions. The enriched EDB-degrading consortium consisted of anaerobic bacteria Desulfovibrio, facultative anaerobe Chromobacterium, and other potential EDB degraders. The results showed that the biodegradation efficiency of EDB was more than 61% at 15 °C, and the EDB biodegradation can be best described by the apparent pseudo-first-order kinetics. EDB biodegradation occurred at a relatively broad range of initial dissolved oxygen (DO) from 1.2 to 5.1 mg/L, indicating that the microbial consortium had a strong ability to adapt. The addition of 40 mg/L of rhamnolipid and 0.3 mM of sodium lactate increased the biodegradation. A two-phase biodegradation scheme was proposed for the EDB biodegradation in this study: an aerobic biodegradation to carbon dioxide and an anaerobic biodegradation via a two-electron transfer pathway of dihaloelimination. To our knowledge, this is the first study that reported EDB biodegradation by an acclimated consortium under both aerobic and anaerobic conditions, a dynamic DO condition often encountered during enhanced biodegradation of EDB in the field.

In situ bioremediation of 1,2-dibromoethane (EDB) in groundwater to part-per-trillion concentrations using cometabolism.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was historically added to leaded gasoline as a scavenger to prevent the build-up of lead oxide deposits in engines. Studies indicate that EDB is present at thousands of past fuel spill sites above its stringent EPA Maximum Contaminant Level (MCL) of 0.05 µg/L. There are currently no proven in situ options to enhance EDB degradation in groundwater to meet this requirement. Based on successful laboratory studies showing that ethane can be used as a primary substrate to stimulate the aerobic, cometabolic biodegradation of EDB to <0.015 µg/L (Hatzinger et al., 2015), a groundwater recirculation system was installed at the FS-12 EDB plume on Joint Base Cape Cod (JBCC), MA to facilitate in situ treatment. Groundwater was taken from an existing extraction well, amended with ethane, oxygen, and inorganic nutrients and then recharged into the aquifer upgradient of the extraction well creating an in situ reactive zone. The concentrations of EDB, ethane, oxygen, and anions in groundwater were measured with time in a series of nested monitoring wells installed between the extraction and injection well. EDB concentrations in the six monitoring wells that were hydraulically well-connected to the pumping system declined from ~ 0.3 µg/L (the average concentration in the recirculation cell after 3 months of operation without amendment addition) to <0.02 µg/L during the 4-month amendment period, meeting both the federal MCL and the more stringent Massachusetts MCL (0.02 µg/L). The data indicate that cometabolic treatment is a promising in situ technology for EDB, and that low regulatory levels can be achieved with this biological approach.

Variable dual carbon-bromine stable isotope fractionation during enzyme-catalyzed reductive dehalogenation of brominated ethenes.

The potential of compound-specific stable isotope analysis (CSIA) to characterize biotransformation of brominated organic compounds (BOCs) was assessed and compared to chlorinated analogues. Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S catalyzed the dehalogenation of tribromoethene (TBE) to either vinyl bromide (VB) or ethene, respectively. Significantly lower isotope fractionation was observed for TBE dehalogenation by S. multivorans (εC = -1.3 ± 0.2‰) compared to D. hafniense (εC = -7.7 ± 1.5‰). However, higher fractionation was observed for dibromoethene (DBE) dehalogenation by S. multivorans (εC = -16.8 ± 1.8‰ and -21.2 ± 1.6‰ for trans- and cis-1,2- (DBE) respectively), compared to D. hafniense PCE-S (εC = -9.5 ± 1.2‰ and -14.5 ± 0.7‰ for trans-1,2-DBE and cis-1,2-DBE, respectively). Significant, but similar, bromine fractionation was observed for for S. multivorans (εBr = -0.53 ± 0.15‰, -1.03 ± 0.26‰, and -1.18 ± 0.13‰ for trans-1,2-DBE, cis-1,2-DBE and TBE, respectively) and D. hafniense PCE-S (εBr = -0.97 ± 0.28‰, -1.16 ± 0.36‰, and -1.34 ± 0.32‰ for cis-1,2-DBE, TBE and trans-1,2-DBE, respectively). Variable CBr dual-element slopes were estimated at Λ (εC/εBr) = 1.03 ± 0.2, 17.9 ± 5.8, and 29.9 ± 11.0 for S. multivorans debrominating TBE, cis-1,2-DBE and trans-1,2-DBE, respectively, and at 7.14 ± 1.6, 8.27 ± 3.7, and 8.92 ± 2.4 for D. hafniense PCE-S debrominating trans-1,2-DBE, TBE and cis-1,2-DBE, respectively. A high variability in isotope fractionation, which was substrate property related, was observed for S. multivorans but not D. hafniense, similar as observed for chlorinated ethenes, and may be due to rate-limiting steps preceding the bond-cleavage or differences in the reaction mechanism. Overall, significant isotope fractionation was observed and, therefore, CSIA can be applied to monitor the fate of brominated ethenes in the environment. Isotope effects differences, however, are not systematically comparable to chlorinated ethenes.

Enhancing aerobic biodegradation of 1,2-dibromoethane in groundwater using ethane or propane and inorganic nutrients.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was previously used as both a soil fumigant and a scavenger in leaded gasoline. EDB has been observed to persist in soils and groundwater, particularly under oxic conditions. The objective of this study was to evaluate options to enhance the aerobic degradation of EDB in groundwater, with a particular focus on possible in situ remediation strategies. Propane gas and ethane gas were observed to significantly stimulate the biodegradation of EDB in microcosms constructed with aquifer solids and groundwater from the FS-12 EDB plume at Joint Base Cape Cod (Cape Cod, MA), but only after inorganic nutrients were added. Ethene gas was also effective, but rates were appreciably slower than for ethane and propane. EDB was reduced to <0.02 µg/L, the Massachusetts state Maximum Contaminant Level (MCL), in microcosms that received ethane gas and inorganic nutrients. An enrichment culture (BE-3R) that grew on ethane or propane gas but not EDB was obtained from the site materials. The degradation of EDB by this culture was inhibited by acetylene gas, suggesting that degradation is catalyzed by a monooxygenase enzyme. The BE-3R culture was also observed to biodegrade 1,2-dichloroethane (DCA), a compound commonly used in conjunction with EDB as a lead scavenger in gasoline. The data suggest that addition of ethane or propane gas with inorganic nutrients may be a viable option to enhance degradation of EDB in groundwater aquifers to below current state or federal MCL values.

Biodegradation of low concentrations of 1,2-dibromoethane in groundwater is enhanced by phenol.

The lead scavenger 1,2-dibromoethane (EDB), a former additive to leaded gasoline, is a common groundwater contaminant, yet not much knowledge is available for its targeted bioremediation, especially under in situ conditions. The study site was an aviation gas spill site, which, although all hydrocarbons and most of the EDB were remediated in the mid-1990s, still exhibits low levels of EDB remaining in the groundwater (about 11 µg EDB/l). To evaluate the effect of phenol on biostimulation of low concentration of EDB, microcosms were established from an EDB-contaminated aquifer. After 300 days at environmentally relevant conditions (12 ± 2 °C, static incubation), EDB was not significantly removed from unamended microcosms compared to the abiotic control. However, in treatments amended with phenol, up to 80 % of the initial EDB concentration had been degraded, while added phenol was removed completely. Microbial community composition in unamended and phenol-amended microcosms remained unchanged, and Polaromonas sp. dominated both types of microcosms, but total bacterial abundance and numbers of the gene for phenol hydroxylase were higher in phenol-amended microcosms. Dehalogenase, an indicator suggesting targeted aerobic biodegradation of EDB, was not detected in either treatment. This finding suggests phenol hydroxylase, rather than a dehalogenation reaction, may be responsible for 1,2-dibromoethane oxidation under in situ conditions. In addition, biostimulation of EDB is possible through the addition of low levels of phenol in aerobic groundwater sites.

Differences in crystallization of two LinB variants from Sphingobium japonicum UT26.

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (µ), and half-saturation coefficients (K(S)) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while µ's were similar for the two compounds, ranging from 0.19 to 0.52 day(-1) with 1,2-DCA to 0.28 to 0.36 day(-1) for EDB. K(S) was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 µg/liter for 1,2-DCA, 0.05 µg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.

Molecular approach to evaluate biostimulation of 1,2-dibromoethane in contaminated groundwater.

This study investigated the effect of co-substrate amendments on EDB biodegradation under aerobic conditions. Microcosms were established using contaminated soil and groundwater samples and maintained under in situ conditions to determine EDB degradation rates, and the diversity and abundance of EDB degrading indigenous bacteria. After 100days of incubation, between 25% and 56% of the initial EDB was degraded in the microcosms, with added jet fuel providing highest degradation rates (2.97±0.49yr(-1)). In all microcosms, the quantity of dehalogenase genes did not change significantly, while the number of BTEX monooxygenase and phenol hydroxylase genes increased with jet fuel amendments. These results indicate that EDB was not degraded by prior dehalogenation, but rather by cometabolism with adapted indigenous microorganisms. This is also reflected in the history of the plume, which originated from an aviation gasoline pipeline leak. This study suggests that biostimulation of EDB is possible at aerobic groundwater sites.

Efficacy of pentane, toluene, and benzene to support aerobic cometabolism of ethylene dibromide.

The ability of pentane, benzene, and toluene to support aerobic cometabolism of ethylene dibromide (1,2-dibromoethane, EDB) was evaluated. A pentane enrichment culture cometabolized EDB, with a transformation capacity of 0.35 µmol EDB/mg biomass (66.2 µg EDB/mg biomass) in the absence of growth substrate. It also cometabolized EDB while actively growing on pentane. However, enrichment cultures grown on benzene or toluene could not cometabolize EDB, with or without their respective growth substrates.

Biodegradation of ethylene dibromide (1,2-dibromoethane [EDB]) in microcosms simulating in situ and biostimulated conditions.

Although 1,2-dibromoethane (EDB) is a common groundwater contaminant, there is the lack of knowledge surrounding EDB biodegradation, especially under aerobic conditions. We have performed an extensive microcosm study to investigate the biodegradation of EDB under simulated in situ and biostimulated conditions. The materials for soil microcosms were collected from an EDB-contaminated aquifer at the Massachusetts Military Reservation in Cape Cod, MA. This EDB plume has persisted for nearly 40 years in both aerobic and anaerobic EDB zones of the aquifer. Microcosms were constructed under environmentally relevant conditions (field EDB and DO concentrations; incubated at 12°C). The results showed that natural attenuation occurred under anaerobic conditions but not under aerobic conditions, explaining why aerobic EDB contamination is so persistent. EDB degradation rates were greater under biostimulated conditions for both the aerobic and anaerobic microcosms. Particularly for aerobic biostimulation, methane-amended microcosms degraded EDB, on average, at a first order rate eight times faster than unamended microcosms. The best performing replicate achieved an EDB degradation rate of 7.0 yr(-1) (half-life (t(1/2))=0.10 yr). Residual methane concentrations and the emergence of methanotrophic bacteria, measured by culture independent bacterial analysis, provided strong indications that EDB degradation in aerobic methane-amended microcosms occurred via cometabolic degradation. These results indicate the potential for enhanced natural attenuation of EDB and that methane could be considered co-substrate for EDB bioremediation for the EDB-contaminated groundwater in aerobic zone.

Screening for reactive metabolites using electro-optical arrays featuring human liver cytosol and microsomal enzyme sources and DNA.

We demonstrate for the first time the combination of human liver cytosol and microsomal enzyme sources into an electro-optical array to screen for reactive metabolites produced in multi-enzyme metabolic processes.

Anaerobic biodegradation of ethylene dibromide and 1,2-dichloroethane in the presence of fuel hydrocarbons.

Field evidence from underground storage tank sites where leaded gasoline leaked indicates the lead scavengers 1,2-dibromoethane (ethylene dibromide, or EDB) and 1,2-dichloroethane (1,2-DCA) may be present in groundwater at levels that pose unacceptable risk. These compounds are seldom tested for at UST sites. Although dehalogenation of EDB and 1,2-DCA is well established, the effect of fuel hydrocarbons on their biodegradability under anaerobic conditions is poorly understood. Microcosms (2 L glass bottles) were prepared with soil and groundwater from a UST site in Clemson, South Carolina, using samples collected from the source (containing residual fuel) and less contaminated downgradient areas. Anaerobic biodegradation of EDB occurred in microcosms simulating natural attenuation, but was more extensive and predictable in treatments biostimulated with lactate. In the downgradient biostimulated microcosms, EDB decreased below its maximum contaminant level (MCL) (0.05 microg/L) at a first order rate of 9.4 +/- 0.2 yr(-1). The pathway for EDB dehalogenation proceeded mainly by dihaloelimination to ethene in the source microcosms, while sequential hydrogenolysis to bromoethane and ethane was predominant in the downgradient treatments. Biodegradation of EDB in the source microcosms was confirmed by carbon specific isotope analysis, with a delta13C enrichment factor of -5.6 per thousand. The highest levels of EDB removal occurred in microcosms that produced the highest amounts of methane. Extensive biodegradation of benzene, ethylbenzene, toluene and ortho-xylene was also observed in the source and downgradient area microcosms. In contrast, biodegradation of 1,2-DCA proceeded at a considerably slower rate than EDB, with no response to lactate additions. The slower biodegradation rates for 1,2-DCA agree with field observations and indicate that even if EDB is removed to below its MCL, 1,2-DCA may persist.

Analysis of DNA adducts formed in vivo in rats and mice from 1,2-dibromoethane, 1,2-dichloroethane, dibromomethane, and dichloromethane using HPLC/accelerator mass spectrometry and relevance to risk estimates.

Dihaloalkanes are of toxicological interest because of their high-volume use in industry and their abilities to cause tumors in rodents, particularly dichloromethane and 1,2-dichloroethane. The brominated analogues are not used as extensively but are known to produce more toxicity in some systems. Rats and mice were treated i.p. with (14)C-dichloromethane, -dibromomethane, -1,2-dichloroethane, or -1,2-dibromoethane [5 mg (kg body weight)(-1)], and livers and kidneys were collected to rapidly isolate DNA. The DNA was digested using a procedure designed to minimize processing time, because some of the potential dihalomethane-derived DNA-glutathione (GSH) adducts are known to be unstable, and the HPLC fractions corresponding to major adduct standards were separated and analyzed for (14)C using accelerator mass spectrometry. The level of liver or kidney S-[2-(N(7)-guanyl)ethyl]GSH in rats treated with 1,2-dibromoethane was approximately 1 adduct/10(5) DNA bases; in male or female mice, the level was approximately one-half of this. The levels of 1,2-dichloroethane adducts were 10-50-fold lower. None of four known (in vitro) GSH-DNA adducts was detected at a level of >2/10(8) DNA bases from dibromomethane or dichloromethane. These results provide parameters for risk assessment of these compounds: DNA binding occurs with 1,2-dichloroethane but is considerably less than from 1,2-dibromoethane in vivo, and low exposure to dihalomethanes does not produce appreciable DNA adduct levels in rat or mouse liver and kidney of the doses used. The results may be used to address issues in human risk assessment.

Effect of alterations of key active site residues in O6-alkylguanine-DNA Alkyltransferase on its ability to modulate the genotoxicity of 1,2-dibromoethane.

The production of mutations and the reduction in survival of cells treated with alpha,omega-dihaloalkanes is greatly enhanced by the presence of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein that removes O6-alkylguanine adducts from DNA [Liu, L., Hachey, D. L., Valadez, G., Williams, K. M., Guengerich, F. P., Loktionova, N. A., Kanugula, S., and Pegg, A. E. (2004) J. Biol. Chem. 279, 4250-4259]. The effects of alterations to key residues in the active site of AGT were studied using AGTs with point mutations. It was found that mutants of AGT at positions Tyr114, Arg128, Pro140, Gly156, Gly160, and Tyr158 did not bring about the increase in genotoxicity of 1,2-dibromoethane seen with wild-type AGT, although these mutants, with the exception of those at Tyr114 and Arg128, are known to have sufficient AGT repair function to be able to protect cells from alkylating agents. The R128A mutant was able to react with 1,2-dibromoethane at the Cys145 acceptor site, but the resulting AGT-Cys145S-(CH2)2Br was much less able to produce a covalent adduct with DNA. This result is explained by the need for AGT to induce a structural change in the DNA "flipping" of a guanine nucleotide into the substrate binding pocket where Cys145 is located since the side chain of residue Arg128 plays a critical role in this reaction. Point mutations in AGT at the other sites (Y114A, P140K, and Y158H) reduced the ability of the protein to react with 1,2-dibromoethane as measured by the loss of activity. These results were confirmed by MS analysis of the tryptic peptide that contains the modified Cys145. There was no change in the stability of the AGT-Cys145S-(CH2)2Br intermediate formed in mutants Y158H and P140K. The reaction was studied in detail with mutant P140K using dihaloalkanes of different length; no effect of the mutations was seen with dibromomethane, but an enhanced difference was observed with 1,3-dibromopropane and 1,5-dibromopentane. These results show that even slight alterations in the active site pocket of AGT that do not prevent its ability to protect cells from alkylating agents can block the paradoxical enhancement of the genotoxicity of the larger alpha,omega-dihaloalkanes by reducing the reaction with Cys145.

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.

Chlorogenate hydrolase-catalyzed synthesis of hydroxycinnamic acid ester derivatives by transesterification, substitution of bromine, and condensation reactions.

A chlorogenate hydrolase (EC 3.1.1.42) synthesized 2-phenylethyl caffeate (2-CAPE) from 5-chlorogenic acid (5-CQA) and 2-phenylethyl alcohol (2-PA) (by transesterification), from 5-CQA and 2-phenylethyl bromide (2-PBr) (by substitution of bromine), and from caffeic acid (CA) and 2-PA or 2-PBr (by condensation) as well as hydrolysis of 5-CQA. Some reaction conditions including pH, temperature, substrate and solvent concentrates, and reaction time were optimized for the production of 2-CAPE. A maximal molar yield of 50% was achieved by transesterification, 4.7% by substitution of bromine, and 13% by condensation. Among the parameters studied for optimization, the pH of the buffer solution and concentration of 2-PA or 2-PBr affected the production of 2-CAPE. The optimum pH for the hydrolysis reaction was within the neutral range (pH 6.5), whereas the residual three reactions were only catalyzed within the acidic range (pH 3.0-4.0). The optimum concentrations of 2-PA and 2-PBr for three reactions were 5-70 vol% and no 2-CAPE was produced in the 2-PA or 2-PBr solutions containing powdered enzyme. The enzyme may bind to the caffeoyl moiety of 5-CQA or CA to form an enzyme-substrate complex. It then catalyzes four different reactions corresponding to the reaction conditions.

Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation.

Mechanisms of toxicity continue to be important in developing rational strategies to deal with chemicals present in the environment. Understanding and predicting toxicity have also become a critical step in the process of drug development. Covalent binding of chemicals to macromolecules is one aspect of toxicity, and the principles and outcomes of the process are considered. Two examples of chemicals for which several aspects of metabolism and reactions are understood are aflatoxin B(1) and polyhalogenated olefins. Ethylene dibromide is a compound that is activated to genotoxic half-mustards by conjugation with glutathione or the DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT). The AGT reaction is unusual, in that crosslinking of the protein to DNA increases mutagenicity. One of the involved mechanisms is formation of N(7)-guanyl crosslinks and depurination to produce G-->T transversions; other reactions appear to yield the additional mutagenic events. The phenomenon of thiol conjugation to increase mutagenicity is widespread among bis-electrophiles.

Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.

It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).

Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase.

Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the dehalogenation pathway. Steady-state kinetic analysis of DhaA with a range of halopropanes showed that bromopropanes had higher k(cat) and lower K(M) values than the chlorinated analogues. The kinetic mechanism of dehalogenation was further studied using rapid-quench-flow analysis of 1,3-dibromopropane conversion. This provided a direct measurement of the chemical steps in the reaction mechanism, i.e., cleavage of the carbon-halogen bond and hydrolysis of the covalent alkyl-enzyme intermediate. The results lead to a minimal mechanism consisting of four main steps. The occurrence of a pre-steady-state burst, both for bromide and 3-bromo-1-propanol, suggests that product release is rate-limiting under steady-state conditions. Combining pre-steady-state burst and single-turnover experiments indicated that the rate of carbon-bromine bond cleavage was indeed more than 100-fold higher than the steady-state k(cat). Product release occurred with a rate constant of 3.9 s(-1), a value close to the experimental k(cat) of 2.7 s(-1). Comparing the kinetic mechanism of DhaA with that of the corresponding enzyme from Xanthobacter autotrophicus GJ10 (DhlA) shows that the overall mechanisms are similar. However, whereas in DhlA the rate of halide release represents the slowest step in the catalytic cycle, our results suggest that in DhaA the release of 3-bromo-1-propanol is the slowest step during 1,3-dibromopropane conversion.