Hydrolytic Dehalogenation Research Articles

4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase,

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

Raman evidence for product binding to the enzyme W137F 4‐chlorobenzoyl‐CoA dehalogenase in two conformational states

AbstractThe enzyme 4‐chlorobenzoyl‐CoA dehalogenase catalyzes the hydrolytic dehalogenation of 4‐chlorobenzoyl‐CoA (4‐CBA‐CoA) to yield 4‐hydroxybenzoyl‐CoA (4‐HBA‐CoA). Part of the catalytic mechanism involves interaction between an active site side‐chain, from Asp145, with the 4‐position of the benzoyl moiety. For the complex consisting of product 4‐HBA‐CoA in the active site the interaction between Asp145 and the 4‐OH group helps produce a major rearrangement in the benzoyl's π‐electron system with the system becoming highly polarized. This leads to a large red shift in the benzoyl λmax, from 260 to 370 nm, and radical changes in the Raman spectrum. In addition to the Asp145‐4‐OH interaction, the aspartate side‐chain is held in place by a hydrogen bond to the indole side‐chain of Trp137. We explored the consequences of removing this hydrogen bonding interaction by using protein engineering techniques to replace Trp137 by Phe, Ala or His. For 4‐HBA‐CoA binding to Trp137Phe dehalogenase there is spectroscopic evidence for two conformational populations for bound product. One species (highly polarized) has a λmax near 375 nm and intense Raman bands at 1550, 1520 and 658 cm−1, whereas the second species (moderately polarized) has a λmax near 330 nm and an intense Raman mode at 1575 cm−1. Raman data collected between 6 and 34 °C demonstrate population changes with the highly polarized species having the highest thermodynamic stability. Copyright © 2005 John Wiley & Sons, Ltd.

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus). After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters. In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity. The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells.

A new dl-2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

dl-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of d- and l-2-haloalkanoic acids to produce the corresponding l- and d-2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for dl-2-haloacid dehalogenase from Pseudomonas putida PP3 ( dl-DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of dl-DEX 312 was carried out in H 2 18 O by use of a large excess of the enzyme with d- or l-2-chloropropionate as a substrate, the lactate produced was labeled with 18 O . This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of l-2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. dl-DEX 312 resembles dl-2-haloacid dehalogenase from Pseudomonas sp. 113 ( dl-DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, dl-DEX 312 is markedly different from dl-DEX 113 in its substrate specificity. We found that dl-DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for dl-DEX 113. dl-DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.

Mass spectrometric analysis of the reactions catalyzed by l-2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

Abstract l -2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of l -2-haloalkanoic acids to produce the corresponding d -2-hydroxyalkanoic acids. Asp10 of l -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.

Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA.

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.

Raman evidence for Meisenheimer complex formation in the hydrolysis reactions of 4-fluorobenzoyl- and 4-nitrobenzoyl-coenzyme A catalyzed by 4-chlorobenzoyl-coenzyme A dehalogenase.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site Asp145 carboxylate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion to form arylated enzyme (EAr). The EAr is then hydrolyzed to product. In this paper, we report the kinetics for dehalogenase-catalyzed 4-fluorobenzoyl-CoA (4-FBA-CoA) and 4-nitrobenzoyl-CoA (4-NBA-CoA) hydrolysis and provide Raman spectral evidence for the accumulation of EMc in these reactions. The 4-FBA-CoA and 4-NBA-CoA substrate analogues were selected for the poor leaving group ability of their C(4) substituents. Thus, the formation of the EAr from EMc should be hindered, giving rise to a quasi-steady-state equilibrium between EMc and the Michaelis complex. Detailed kinetic studies were carried out to quantitate the composition of the reaction mixtures. Quench experiments demonstrated that significant populations of EAr do not exist in reaction mixtures involving the 4-F- or 4-N-substrates. A kinetic model enabled us to estimate that approximately 10-20% of the enzyme-substrate complexes in the reaction mixtures are present as EMc. Raman difference spectra of 4-NBA-CoA and 4-FBA-CoA bound to WT and H90Q mutant dehalogenase have broad features near 1500 and 1220 cm(-1) that are absent in the free ligand. Crucially, these features are also absent in the Raman spectra of the complexes involving the D145A dehalogenase mutant that are unable to form an EMc. Quantum mechanical calculations, at the DFT level, provide strong support for assigning the novel 1500 and 1220 cm(-1) features to an EMc.

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment.

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.

Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. This study examines the role of binding interactions in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-CoA complex. These groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interaction. The changes in the Gibbs free energy of the enzyme-substrate complex (DeltaDeltaG(ES)) and enzyme-transition state complex (DeltaDeltaG) brought about by the modification were measured. Cases where DeltaDeltaG exceeds DeltaDeltaG(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis, we show that the H-bond interactions between the Gly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducing the energy barrier to catalysis by an additional 3.3 kcal/mol. Together, these binding interactions contribute approximately 10(6) to the k(cat)/K(m).

Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis.

4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C4 of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 microM(-1) x s(-1)) and 4-HBA-CoA (33 and 11 microM(-1) x s(-1)) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) and 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and mutant dehalogenase are approximately 200 s(-1) while the rate constants for EAr formation are 40 and 0.3 s(-1), respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.

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.

Novel Catalytic Mechanism of Nucleophilic Substitution by Asparagine Residue Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction

l-2-Haloacid dehalogenase from Pseudomonas sp. YL catalyzes the hydrolytic dehalogenation, in which Asp(10) acts as a nucleophile to attack the alpha-carbon of l-2-haloalkanoates to form an ester intermediate, which is subsequently hydrolyzed to produce d-2-hydroxyalkanoates. Surprisingly, replacement of the catalytic residue, Asp(10), by Asn did not result in total inactivation of the enzyme (Kurihara, T., Liu, J.-Q., Nardi-Dei, V., Koshikawa, H., Esaki, N., and Soda, K. (1995) J. Biochem. 117, 1317-1322). In this study, we monitored the D10N mutant enzyme reaction by ion-spray mass spectrometry, and found that the enzyme shows a unique structural change when it was incubated with the substrate, l-2-chloropropionate. LC/MS and tandem MS/MS analyses revealed that Asn(10) attacks the substrate to form an imidate, and a proton and d-lactic acid are eliminated to produce a nitrile (beta-cyanoalanine residue), followed by hydrolysis to reproduce Asn(10). This is the first report of the function of Asn to catalyze nucleophilic substitution through its conversion to beta-cyanoalanine residue as an intermediate structure. Also, these results demonstrate that mass spectrometry is remarkably useful in monitoring enzyme reactions.

Cloning and expression of DL-2-haloacid dehalogenase gene from Burkholderia cepacia

Burkholderia cepacia strain KY, which can utilize a herbicide 2,4-D as a sole carbon and energy source, catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids. We have cloned the gene encoding DL-2-haloacid dehalogenase, and obtained a recombinant plasmid (pUCDEXL) containing approximately 4.5 kbp insert. In both of B. cepacia strain KY and this clone E. coli JM109/pUCDEXL, DL-2-haloacid dehalogenase was induced significantly with monohalogenated acetic acids, such as chloroacetate, bromoacetate and iodoacetate. This dehalogenase was also overexpressed in E. coli using three different promoters. In pET vector systems with T7 lac promoter, a large amount of dehalogenase was selectively expressed, but some parts of the protein were accumulated in the form of inclusion bodies. This problem was overcome to carry on growth and induction at 22°C, and at the same time, the maximum specific activity of dehalogenase was reached at 12.6 U/mg, 500-fold higher activity than in wild strain, B. cepacia strain KY grown with 2,4-D.

Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria.

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.

Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by l-2-Haloacid Dehalogenase

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8).

Dl-2-Haloacid Dehalogenase fromPseudomonas sp. 113 Is a New Class of Dehalogenase Catalyzing Hydrolytic Dehalogenation Not Involving Enzyme-Substrate Ester Intermediate

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.

Monooxygenase-mediated 1,2-dichloroethane degradation by Pseudomonas sp. strain DCA1.

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a Km value below the detection limit of 0.5 microM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.

Mechanism-based Quantitative Structure–Biodegradability Relationships for Hydrolytic Dehalogenation of Chloro- and Bromo-Alkenes

Quantitative Structure–Biodegradability Relationships (QSBRs) have been developed for microbial hydrolytic dehalogenation of small chlorinated and brominated alkenes. Two different reaction mechanisms, S2 and S2′, were considered for the dehalogenation of small haloalkenes by hydrolytic dehalogenases. Multivariate QSBRs based on quantum-chemically calculated descriptors were used to distinguish between these dehalogenation mechanisms. A QSBR based on atom valence and partial atomic charge on electrophilic site, together with the energy of the lowest unoccupied molecular orbital, molecular weight and dipole moment were able to explain 92% (81% cross-validated) of the quantitative variance in the dehalogenation rates. This QSBR supported a S2 reaction mechanism. This reaction mechanism was also confirmed by GC-MS identification of the reaction products for the compounds which should give discriminating reaction products depending on mechanism. The substrate specificity of hydrolytic dehalogenases for unsaturated halogenated substrates is discussed in the light of chemical structure and properties of the substrate molecules.

Mechanism-based Quantitative Structure–Biodegradability Relationships for Hydrolytic Dehalogenation of Chloro- and Bromo-Alkenes

Mechanism-based Quantitative Structure–Biodegradability Relationships for Hydrolytic Dehalogenation of Chloro- and Bromo-Alkenes