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

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. 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 -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the k_cat values of the enzymes of these strains are 0.6 and 3.3 s⁻¹, respectively, and the K_m values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the K_m of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.     

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH₂-dependent manner.     

Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg²⁺ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 °C. The apparent K _m value for 4-chlorobenzoyl-coenzyme A was 2.4–2.7 µM. V _max was 1.1 × 10^–7 M sec^–1 (2.2 µmol min^–1 mg^–1 of protein). The NH₂-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.Abbreviations CBA chlorobenzoate - CoA coenzyme A - HBA hydroxybenzoate - DTT dithiothreitol - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis     

Degradation and Fate of Carbon Tetrachloride in Unadapted Methanogenic Granular Sludge

The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 μM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 μmol of CT g of volatile suspended solids⁻¹ day⁻¹. The main end products of CT degradation were CO₂ and Cl⁻, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [¹⁴C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS₂. The amount of CO₂ produced (23%) was lower and the amount of Cl⁻ produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO₂. Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.     

Biodegradation of chlorinated paraffins and long-chain chloroalkanes by Rhodococcus sp. S45-1

A strain of the genus Rhodococcus, designated isolate S45-1, was isolated from an environmental water sample by enrichment, using the chlorinated paraffin Cereclor S45 as the sole carbon and energy source. This is the first report of microbial utilisation of chlorinated paraffins as sole source of carbon and energy. Biochemical studies of isolate S45-1 revealed little similarity with other Rhodococcus species. Isolate S45-1 was able to utilise 1-chloroalkanes of chain-length 12–18C as sole source of carbon and energy. Gas chromatography-mass spectrometry of the reaction medium indicated that γ-butyrolactone was formed as a product of 1-chlorotetradecane metabolism.     

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

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

The 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 K_M was 10.96 mM when treated with dichloromethane as substrate. The fitted curve of dichloromethane degradation gave a V_max 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.     

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.

     

Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in gamma-HCH degradation

Aim: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)‐isomers based on dechlorinase enzyme activity and characterize genes and metabolites. Methods and Results: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH‐degrading activity. A bacterium able to grow on α‐, β‐, γ‐ and δ‐HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. γ‐Pentachlorocyclohexene 1,2,4‐trichlorobenzene and chlorohydroquinone were identified as metabolites. Conclusions: The study demonstrates that HCH‐degrading bacteria can be identified from large environmental sample‐based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high‐throughput manner to screen bacteria for chlorinated compounds. Significance and Impact of the Study: The chlorinated pesticide HCH is a persistent and toxic environmental pollutant which needs to be remediated. Isolation of diverse bacterial species capable of degrading all the isomers of HCH will help in large‐scale bioremediation in various parts of the world.     

Dichloromethane as the sole carbon source forHyphomicrobium sp. strain DM2 under denitrification conditions

Hyphomicrobium sp. strain DM2 was found to grow anaerobically in the presence of nitrate with methanol, formaldehyde, formate or dichloromethane. The estimated growth rate constants with methanol and dichloromethane under denitrification conditions were 0.04 h^–1 and 0.015 h^–1, respectively, which is twofold and fourfold lower than the rates of aerobic growth with these substrates. Slight accumulation of nitrite was observed in all cultures grown anaerobically with nitrate. Dichloromethane dehalogenase, the key enzyme in the utilization of this carbon source, was induced under denitrification conditions to the same specific activity level as under aerobic conditions. In a fed batch culture under denitrification conditionsHyphomicrobium sp. DM2 cumulatively degraded 35 mM dichloromethane within 24 days. This corresponds to a volumetric degradation rate of 5 mg dichloromethane/l·h and demonstrates that denitrificative degradation offers an attractive possibility for the development of anaerobic treatment systems to remove dichloromethane from contaminated groundwater.     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Metabolism of homoacetogens

Homoacetogenic bacteria are strictly anaerobic microorganisms that catalyze the formation of acetate from C₁ units in their energy metabolism. Most of these organisms are able to grow at the expense of hydrogen plus CO₂ as the sole energy source. Hydrogen then serves as the electron donor for CO₂ reduction to acetate. The methyl group of acetate is formed from CO₂ via formate and reduced C₁ intermediates bound to tetrahydrofolate. The carboxyl group is derived from carbon monoxide, which is synthesized from CO₂ by carbon monoxide dehydrogenase. The latter enzyme also catalyzes the formation of acetyl-CoA from the methyl group plus CO. Acetyl-CoA is then converted either to acetate in the catabolism or to cell carbon in the anabolism of the bacteria. The homoacetogens are very versatile anaerobes, which convert a variety of different substrates to acetate as the major end product.     

Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts

Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione S-transferase was demonstrated. Adducts included dichloromethane carbon and glutathione sulfur atoms. A reaction with DNA occurred preferentially at guanine bases. Increased DNA degradation in a polA mutant of Methylobacterium dichloromethanicum DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.     

Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S-transferase theta 1-1 variants.

Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.     

Purification and characterization of a lyase from the EDTA-degrading bacterial strain DSM 9103 that catalyzes the splitting of [S,S]-ethylenediaminedisuccinate, a structural isomer of EDTA

The bacterial strain DSM 9103, able to utilize EDTA as a sole source of carbon, nitrogen, and energy, is also capable to grow with [S,S]-ethylenediaminedisuccinate ([S,S]-EDDS), a structural isomer of EDTA. In cell-free extracts of[S,S]-EDDS-grown bacteria, [S,S]-EDDS degradation was observed in the absence of any cofactors. An enzyme was purified41-fold that catalyzed the non-hydrolytic splitting of[S,S]-EDDS leading to the formation of fumarate and N-(2-aminoethyl) aspartic acid. These data strongly suggest that the enzyme belongs to the group of carbon-nitrogen lyases. The splitting reaction was reversible, and an equilibrium constant of approximately 43.0 10^-1 M was determined. Out of the three stereo-isomers of EDDS, [S,S]-and [R,S]-EDDS were accepted as substrates by the lyase,whereas [R,R]-EDDS remained unchanged in assays with both cell-free extracts and pure enzyme. The enzyme catalyzed the transformation of free [S,S]-EDDS and of [S,S]-EDDS-metal complexes with stability constant lower than 10, namely of MgEDDS, CaEDDS, BaEDDS and to a small extent also of MnEDDS;Fe_IIIEDDS, NiEDDS, CuEDDS, CoEDDS and ZnEDDS were not transformed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Malonate added to peptone-meat extract medium has been shown to induce maleate cis-trans isomerase in Alcaligenes faecalis IB-l4. This enzyme played an indispensable role in the enzymatic production of L-aspartic acid from maleic acid and ammonia.

Though malonate in the medium inhibited the growth of A. faecalis IB-14 to some extent, cells grown in the medium containing 10^-1 M of malonate showed the highest level of the enzyme activity. Specific activity of the enzyme of malonate-grown cells was approximately ten times as strong as that of maleate-grown cells, while, in basal mediumgrown cells, cis-trans isomerization of maleate did not occur at all. Maleate was utilized not only as carbon source of the cell growth but also as inducer for the formation of maleate cis-trans isomerase. On the other hand, malonate was not utilized as carbon source and the metabolism of it within the cell was rather restricted within narrow limits. Thus it was concluded that malonate was a gratuitous inducer for the formation of the enzyme, while maleate, which was considerably metabolized, was normal inducer.

It was demonstrated that most of radioactivity of l-C¹⁴-malonate taken up by cells was localized in particle fraction which sedimented at 105,000×g for 120 minutes, whereas radioactivity of 1-4-C¹⁴-maleate was uniformly distributed in both particulate and soluble fractions. Maleate cis-trans isomerase activity, in turn, was detected exclusively in soluble fraction in both malonate and maleate induced cells.     

The Biology of Methylobacteria Capable of Degrading Halomethanes

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyltetrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH₂O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is a quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and the protection of the biosphere from these toxic pollutants.     

Anaerobic dissimilatory phosphite oxidation,an extremely efficient concept of microbial electron economy

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (−690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD⁺-dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO₂ fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid