Genetics and Biochemistry of Dehalogenating Enzymes

Janssen, Dick B.; Pries, Frens; Ploeg, Jan R. van der

doi:10.1146/annurev.mi.48.100194.001115

Cited by 179 publications

(51 citation statements)

References 78 publications

(21 reference statements)

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“…Thereafter, 150 l of 100 mM Tris-H 2 SO 4 (pH 7.5) containing 5 g of trypsin prepared with H 2 18 O was added to this solution and incubated at 37°C for 12 h.…”

Section: Materials-hmentioning

confidence: 99%

“…Various enzymes catalyzing hydrolytic dehalogenation of organohalogen compounds have been isolated and characterized (1)(2)(3). These enzymes include 2-haloacid dehalogenases (EC 3.8.1.2), haloacetate dehalogenases (EC 3.8.1.3), haloalkane dehalogenases (EC 3.8.1.5), and 4-chlorobenzoyl-CoA dehalogenases (EC 3.8.1.6).…”

mentioning

confidence: 99%

“…O, 10 nmol of DL-DEX 113, 1.0 mol of D-or L-2-chloropropionate (neutralized with NaOH), and 20 mol of Tris-H 2 SO 4 (pH 9.5) were mixed in 50 l of H 2 18 O and incubated at 37°C for 24 h. A 20-l aliquot was lyophilized and dissolved with 20 l of H 2 18 O containing 8 M urea. After incubation at 37°C for 1 h, 30 l of 1 M Tris-H 2 SO 4 (pH 9.0) in H 2 18 O and 10 l of 33 M lysyl endopeptidase in H 2 18 O were added to this solution, and incubation was carried out at 37°C for 12 h.…”

mentioning

confidence: 99%

“…After incubation at 37°C for 1 h, 30 l of 1 M Tris-H 2 SO 4 (pH 9.0) in H 2 18 O and 10 l of 33 M lysyl endopeptidase in H 2 18 O were added to this solution, and incubation was carried out at 37°C for 12 h.…”

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confidence: 99%

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

Nardi‐Dei¹,

Kurihara²,

Park³

et al. 1999

Journal of Biological Chemistry

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18 O of the solvent directly attacks the ␣-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.Various enzymes catalyzing hydrolytic dehalogenation of organohalogen compounds have been isolated and characterized (1-3). These enzymes include 2-haloacid dehalogenases (EC 3.8.1.2), haloacetate dehalogenases (EC 3.8.1.3), haloalkane dehalogenases (EC 3.8.1.5), and 4-chlorobenzoyl-CoA dehalogenases (EC 3.8.1.6). 2-Haloacid dehalogenases are further classified into three groups based on their substrate specificities (4). L-2-Haloacid dehalogenase (L-DEX) 1 specifically acts on L-2-haloalkanoic acids, and the corresponding D-2-hydroxyalkanoic acids are produced. D-2-Haloacid dehalogenase (D-DEX) catalyzes the conversion of D-2-hydroxyalkanoic acids into L-2-hydroxyalkanoic acids. DL-2-Haloacid dehalogenase (DL-DEX) dehalogenates both D-and L-2-haloalkanoic acids, and the corresponding L-and D-2-hydroxyalkanoic acids are produced. DL-DEX is similar to racemases and epimerases in that it acts indiscriminately on the chiral center of both D-and L-enantiomers. However, this enzyme is unique in that it catalyzes a chemical conversion on the chiral centers of both enantiomers. Thus far, the reaction mechanisms of L-DEX from Pseudomonas sp. YL (L-DEX YL) (5-7), haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (8 -10), and 4-chlorobenzoylCoA dehalogenases from Pseudomonas sp. strain CBS3 (11,12) and Arthrobacter sp. 4-CB1 (13) have been analyzed. Their reactions proceed as shown in Fig. 1A. Each of these dehalogenases has an acidic amino acid residue whose carboxylate group attacks the carbon atom of the substrate to which the halogen atom is bound. Asp 10 of L-DEX YL, Asp 124 of haloalkane dehalogenase from X. autotrophicus GJ10, and Asp 145 of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS3 were identified to play this essential role for respective enzymes. The ester intermediates produced in the course of these reactions are subsequently hydrolyzed releasing the products and restoring the carboxylate groups of the enzymes. These were confirmed by chemical modification, sitedirected mutagenesis, mass spectrometry, and x-ray crystallographical analysis (5-13).DL-DEXs have been purified from Pseudomonas sp. 113 (DL-DEX 113) (14), Pseudomonas putida PP3 (15), and Rhizobium sp. (16). However, none of the reaction mechanisms of these DL-DEXs have been studied, and it is unknown whether the reaction mechanism of DL-DEX is similar to that of other halidohydrolases (dehalogenases that catalyze the hydrolytic dehalogenation). We previously determined the primary structure of DL-DEX 113 (Fig. 2), and found that it is similar to that of D-DEX from Pseudomonas putida AJ1 (17). We also showed that DL-DEX 113 has a single and common catalytic site for both D-and L-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17). In the present study, we analyze...

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“…Thereafter, 150 l of 100 mM Tris-H 2 SO 4 (pH 7.5) containing 5 g of trypsin prepared with H 2 18 O was added to this solution and incubated at 37°C for 12 h.…”

Section: Materials-hmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Nardi‐Dei¹,

Kurihara²,

Park³

et al. 1999

Journal of Biological Chemistry

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“…L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids (2)(3)(4). Our recent 18 O incorporation experiment showed that the reaction of L-2-haloacid dehalogenase from Pseudomonas sp.…”

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confidence: 99%

Paracatalytic Inactivation of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL by Hydroxylamine

劉

Kurihara

Miyagi

et al. 1997

Journal of Biological Chemistry

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Asp 10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the ␣-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309 -18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly 6 -Lys 11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp 10 . When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate ␤-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.Paracatalytic enzyme modification is a catalysis-linked and substrate-dependent enzyme modification (1). It involves a direct chemical reaction between an enzyme-activated substrate and an extrinsic reagent. The catalytic effect of an enzyme can increase the reactivity of a substrate with extrinsic reagents that are not constituents of the normal enzyme-substrate system. The reactive intermediates formed may thus react with extrinsic reagents to branch off from the normal catalytic pathway. Consequently, the enzyme active site may be specifically and irreversibly modified.L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids (2-4). Our recent 18 O incorporation experiment showed that the reaction of L-2-haloacid dehalogenase from Pseudomonas sp. YL (L-DEX YL) 1 proceeds through the mechanism shown in Fig. 1 (11) and found that L-DEX YL is paracatalytically inactivated by hydroxylamine. Tandem MS/MS spectrometric analysis revealed that the active site Asp 10 was specifically labeled. Hydroxylamine is thus useful to probe the active site carboxylate group, which constitutes an enzyme-substrate ester intermediate. EXPERIMENTAL PROCEDURES MaterialsLeu 11 , Ser 176 , and Arg 185 were replaced by Lys by site-directed mutagenesis, and the resultant mutant enzyme, L-DEX T15, yields a small peptide fragment containing active site Asp 10 of L-DEX YL by lysyl endopeptidase digestion. (5). Catalytic properties of L-DEX T15 such as the specific activity for L-2-chloropropionate an...

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Bioremediation

Prince¹

2000

Kirk-Othmer Encyclopedia of Chemical Technology

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Bioremediation seeks to use biological processes to restore contaminated environments to productive use. Many contaminants at industrial, agricultural, and accident sites are amenable to bioremediation. Most organic contaminants are biodegradable under some conditions, and optimizing this biodegradation is the principal thrust of bioremediation to date. Widely used treatments for hydrocarbons include the addition of oxygen or alternative electron acceptors, and the provision of limiting nutrients such as nitrogen and phosphorus. More recalcitrant organic contaminants, such as some halogenated products and some pesticides, herbicides and explosives, require more sophisticated approaches. These have included the addition of co‐substrates to stimulate the growth of degrading organisms, various treatments to increase the bioavailability of the contaminants, and sequential anaerobic and aerobic degradation. Inorganic contaminants are also amenable to bioremediation, since they may be sequestered or extracted from soils and waters by microbes and plants. Bioremediation competes in the marketplace with a range of chemical and physical treatments for cleaning contaminated sites and waters; in general bioremediation is among the least expensive and most benign, but also one of the slowest options.

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Genetics and Biochemistry of Dehalogenating Enzymes

Cited by 179 publications

References 78 publications

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

Paracatalytic Inactivation of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL by Hydroxylamine

Bioremediation

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