Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

Liu, Jiquan; Kurihara, Tatsuo; Nardi‐Dei, Vincenzo; Okamura, Tokumitsu; Esaki, Nobuyoshi; Soda, Kenji

doi:10.1007/bf00700461

Cited by 10 publications

(5 citation statements)

References 14 publications

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“…113, L-DEX YL (19), and L-DEX from Pseudomonas putida no. 109 (20) (final 0.45 mg/ml) were mixed with 1 M hydroxylamine in 1 M Tris-H 2 SO 4 (pH 9.0) in the presence or absence of 100 mM L-2-chloropropionate and incubated at 30°C for 60 min.…”

Section: Reaction Mechanism Of Dl-2-haloacid Dehalogenase 20978mentioning

confidence: 99%

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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Section: Reaction Mechanism Of Dl-2-haloacid Dehalogenase 20978mentioning

confidence: 99%

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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“…The enzyme (L-DEX YL) has been purified to homogeneity and characterized physicochemically (1). Its structural gene has been cloned and overexpressed in Escherichia coli (8,11). L-DEX YL is a dimeric enzyme (1).…”

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Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL

Takaya

Hata

Fujii

et al. 1996

Journal of Biological Chemistry

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114

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L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

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“…Although expression vectors have been used for the overproduction of a l ‐haloacid dehalogenase from Pseudomonas sp. YL (20), to our knowledge there have not been previous reports on the modification of dehalogenases for purification—immobilization purposes. There are few examples of enzymes engineered for immobilization purposes.…”

Section: Discussionmentioning

confidence: 95%

Covalent and Metal-Chelate Immobilization of a Modified 2-Haloacid Dehalogenase for the Enzymatic Resolution of Optically Active Chloropropionic Acid

et al. 2000

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The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively.

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Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

Cited by 10 publications

References 14 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

Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL

Covalent and Metal-Chelate Immobilization of a Modified 2-Haloacid Dehalogenase for the Enzymatic Resolution of Optically Active Chloropropionic Acid

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