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Jennifer A. Littlechild - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of an l-Haloacid Dehalogenase from the Marine Psychrophile Psychromonas ingrahamii with Potential Industrial Application
Marine Biotechnology, 2013Co-Authors: Halina Rose Novak, Christopher Sayer, Jana Panning, Jennifer A. LittlechildAbstract:The recombinant l -Haloacid dehalogenase from the marine bacterium Psychromonas ingrahamii has been cloned and over-expressed in Escherichia coli . It shows activity towards monobromoacetic (100 %), monochloroacetic acid (62 %), S -chloropropionic acid (42 %), S -bromopropionic acid (31 %), dichloroacetic acid (28 %) and 2-chlorobutyric acid (10 %), respectively. The l -Haloacid dehalogenase has highest activity towards substrates with shorter carbon chain lengths (≤C3), without preference towards a chlorine or bromine at the α-carbon position. Despite being isolated from a psychrophilic bacterium, the enzyme has mesophilic properties with an optimal temperature for activity of 45 °C. It retains above 70 % of its activity after being incubated at 65 °C for 90 min before being assayed at 25 °C. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. The V _max and K _m were calculated to be 0.6 μM min^−1 mg^−1 and 1.36 mM with monobromoacetic acid, respectively. This solvent-resistant and stable l -Haloacid dehalogenase from P . ingrahamii has potential to be used as a biocatalyst in industrial processes.
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marine rhodobacteraceae l Haloacid dehalogenase contains a novel his glu dyad that could activate the catalytic water
FEBS Journal, 2013Co-Authors: Halina Rose Novak, Michail N. Isupov, Am Spragg, C. Sayer, Konrad Paszkiewicz, D. Gotz, Jennifer A. LittlechildAbstract:The putative l-Haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an l-Haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 μm·min−1·mg−1 of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied l-Haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other l-Haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 A from its position in other l-Haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the l-Haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst. Database The coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 2yml, 2ymm, 2ymp, 2ymq and 2yn4. Nucleotide sequence data has been deposited in the GenBank database under the accession number JX868516.
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Marine Rhodobacteraceae L-Haloacid Dehalogenase Contains a Novel His/Glu Dyad that Could Activate the Catalytic Water.
The FEBS journal, 2013Co-Authors: Halina Rose Novak, Michail N. Isupov, Am Spragg, C. Sayer, Konrad Paszkiewicz, D. Gotz, Jennifer A. LittlechildAbstract:The putative l-Haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an l-Haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 μm·min−1·mg−1 of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied l-Haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other l-Haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 A from its position in other l-Haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the l-Haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst. Database The coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 2yml, 2ymm, 2ymp, 2ymq and 2yn4. Nucleotide sequence data has been deposited in the GenBank database under the accession number JX868516.
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biochemical and structural studies of a l Haloacid dehalogenase from the thermophilic archaeon sulfolobus tokodaii
Extremophiles, 2009Co-Authors: Michail N. Isupov, Andrey Lebedev, Jennifer A. LittlechildAbstract:Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-Haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-Haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays Haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.
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An order–disorder twin crystal of l‐2‐Haloacid dehalogenase from Sulfolobus tokodaii
Acta Crystallographica Section D-biological Crystallography, 2007Co-Authors: Michail N. Isupov, Andrey Lebedev, Jennifer A. LittlechildAbstract:The l-2-Haloacid dehalogenase enzymes catalyse the hydrolytic cleavage of a halogen from the C2 position of short-chain Haloacids. The recombinant dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned, overexpressed and purified to homogeneity. The 24 kDa enzyme was crystallized using the microbatch method in the monoclinic space group C2, with unit-cell parameters a = 127.6, b = 58.1, c = 51.2 A, β = 97.2°. Data were collected to 1.9 A resolution using synchrotron radiation and the structure was solved by molecular replacement. Analysis of the data and the preliminary refined model showed that the crystal was an order–disorder twin by reticular merohedry with a twin index of 10. It was possible to detwin the experimental data utilizing the symmetry of the molecular layers from which the crystal is built.
Kenji Soda - One of the best experts on this subject based on the ideXlab platform.
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Bacterial 2-Haloacid dehalogenases: Structures and catalytic properties
2015Co-Authors: Kenji Soda, Tatsuo Kurihara, Ji Quan Liu, Vincenzo Nardi-dei, Chung Park, Susumu Tsunasawa, Masaru Myagi, Nobuyoshi EsahAbstract:*To whom correspondence should be addressed Abstract: 2-Haloacid dehalogenases (2-Haloacid halidohydrolase; EC class: 3.8.1.2) catalyze the hydrolytlc dehalogenation of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids. Four different groups of 2-Haloacid dehalogenases have been found in bacterial cells. The carboxylate group of Asplo of L-2-Haloacid dehalogenase acts as a nucleophile on the a-carbon of L-Zhaloalkanoic acid to form an ester intermdate, which is hydrolyzed to produce the corresponding 2-hydroxyalkanoic acid. In contrast, in the reaction of DL-ZHaloacid dehalogenase (inversion type), a water molecule activated by the enzyme directly attacks the a-carbon of the substrate. D-2-Haloacid dehalogenase shows sequence similarity to DL-ZHaloacid dehalogenase (inversion type), suggesting that the reaction mechanism of D-ZHaloacid dehalogenase is similar to that of DL-?-Haloacid dehalogenase (inversion type). Only the DL-2-Haloacid dehalogenase (retention type) reaction proceeds with retention of the C2-configuration of the substrate, and its reaction mechanism is probably different from those of other 2-Haloacid dehalogenases
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A new dl-2-Haloacid dehalogenase acting on 2-Haloacid amides: purification, characterization, and mechanism
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Chung Park, Kenji Soda, Tatsuo Kurihara, Tohru Yoshimura, Nobuyoshi EsakiAbstract:Abstract 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.
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Bacterial 2-Haloacid dehalogenases: structures and reaction mechanisms
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Tatsuo Kurihara, Nobuyoshi Esaki, Kenji SodaAbstract:Abstract Microbial dehalogenases have been attracting a great deal of attention because of their possible application to fine chemical synthesis and bioremediation of halo compound-polluted environment. Dehalogenases employ various different mechanisms to cleave the carbon–halogen bond. 2-Haloacid dehalogenases catalyze the hydrolytic dehalogenation of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids. The reaction mechanism of l -2-Haloacid dehalogenase from Pseudomonas sp. YL has been clarified by 18O incorporation experiment, site-directed mutagenesis and X-ray crystallographic analysis. The carboxylate group of Asp10 performs a nucleophilic attack on the α-carbon atom of the substrate to displace the halogen atom and produce the ester intermediate, which is subsequently hydrolyzed to produce the corresponding d -2-hydroxyalkanoic acid and regenerate the Asp10 residue. The reaction catalyzed by fluoroacetate dehalogenase from Moraxella sp. B similarly proceeds in two steps: the carboxylate group of Asp105 performs a nucleophilic attack on the substrate α-carbon atom to form an ester intermediate, and the intermediate is hydrolyzed by a water molecule activated by His272. In contrast with these two enzymes, a water molecule directly attacks the substrate to displace the halogen atom and produce 2-hydroxyalkanoic acid in the reaction catalyzed by dl -2-Haloacid dehalogenase from Pseudomonas sp. 113.
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dl 2 Haloacid dehalogenase from pseudomonas sp 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme substrate ester intermediate
Journal of Biological Chemistry, 1999Co-Authors: Vincenzo Nardidei, Kenji Soda, Tatsuo Kurihara, Chung Park, Masaru Miyagi, Susumu Tsunasawa, Nobuyoshi EsakiAbstract:Abstract dl-2-Haloacid dehalogenase fromPseudomonas sp. 113 (dl-DEX 113) catalyzes the hydrolytic dehalogenation of d- andl-2-haloalkanoic acids, producing the correspondingl- 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 H2 18O with a 10 times smaller amount of the substrate, either d- or l-2-chloropropionate, the major product was found to be18O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H2 18O, 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 18O. These results indicate that the H2 18O of the solvent directly attacks the α-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.
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Bacterial DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-Haloacid dehalogenases.
Journal of bacteriology, 1997Co-Authors: Vincenzo Nardi-dei, Tatsuo Kurihara, Chung Park, Nobuyoshi Esaki, Kenji SodaAbstract:DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-Haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-Haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-Haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-Haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.
Nobuyoshi Esaki - One of the best experts on this subject based on the ideXlab platform.
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Binding modes of DL-2-Haloacid dehalogenase revealed by crystallography, modeling and isotope effects studies.
Archives of Biochemistry and Biophysics, 2013Co-Authors: Agata Siwek, Tatsuo Kurihara, Nobuyoshi Esaki, Rie Omi, Ken Hirotsu, Keiji Jitsumori, Piotr PanethAbstract:Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-Haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step.
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A new dl-2-Haloacid dehalogenase acting on 2-Haloacid amides: purification, characterization, and mechanism
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Chung Park, Kenji Soda, Tatsuo Kurihara, Tohru Yoshimura, Nobuyoshi EsakiAbstract:Abstract 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.
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Mass spectrometric analysis of the reactions catalyzed by l-2-Haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tatsuo Kurihara, Masaru Miyagi, Susumu Tsunasawa, Susumu Ichiyama, Nobuyoshi EsakiAbstract: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.
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Bacterial 2-Haloacid dehalogenases: structures and reaction mechanisms
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Tatsuo Kurihara, Nobuyoshi Esaki, Kenji SodaAbstract:Abstract Microbial dehalogenases have been attracting a great deal of attention because of their possible application to fine chemical synthesis and bioremediation of halo compound-polluted environment. Dehalogenases employ various different mechanisms to cleave the carbon–halogen bond. 2-Haloacid dehalogenases catalyze the hydrolytic dehalogenation of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids. The reaction mechanism of l -2-Haloacid dehalogenase from Pseudomonas sp. YL has been clarified by 18O incorporation experiment, site-directed mutagenesis and X-ray crystallographic analysis. The carboxylate group of Asp10 performs a nucleophilic attack on the α-carbon atom of the substrate to displace the halogen atom and produce the ester intermediate, which is subsequently hydrolyzed to produce the corresponding d -2-hydroxyalkanoic acid and regenerate the Asp10 residue. The reaction catalyzed by fluoroacetate dehalogenase from Moraxella sp. B similarly proceeds in two steps: the carboxylate group of Asp105 performs a nucleophilic attack on the substrate α-carbon atom to form an ester intermediate, and the intermediate is hydrolyzed by a water molecule activated by His272. In contrast with these two enzymes, a water molecule directly attacks the substrate to displace the halogen atom and produce 2-hydroxyalkanoic acid in the reaction catalyzed by dl -2-Haloacid dehalogenase from Pseudomonas sp. 113.
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dl 2 Haloacid dehalogenase from pseudomonas sp 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme substrate ester intermediate
Journal of Biological Chemistry, 1999Co-Authors: Vincenzo Nardidei, Kenji Soda, Tatsuo Kurihara, Chung Park, Masaru Miyagi, Susumu Tsunasawa, Nobuyoshi EsakiAbstract:Abstract dl-2-Haloacid dehalogenase fromPseudomonas sp. 113 (dl-DEX 113) catalyzes the hydrolytic dehalogenation of d- andl-2-haloalkanoic acids, producing the correspondingl- 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 H2 18O with a 10 times smaller amount of the substrate, either d- or l-2-chloropropionate, the major product was found to be18O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H2 18O, 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 18O. These results indicate that the H2 18O of the solvent directly attacks the α-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.
Tatsuo Kurihara - One of the best experts on this subject based on the ideXlab platform.
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Bacterial 2-Haloacid dehalogenases: Structures and catalytic properties
2015Co-Authors: Kenji Soda, Tatsuo Kurihara, Ji Quan Liu, Vincenzo Nardi-dei, Chung Park, Susumu Tsunasawa, Masaru Myagi, Nobuyoshi EsahAbstract:*To whom correspondence should be addressed Abstract: 2-Haloacid dehalogenases (2-Haloacid halidohydrolase; EC class: 3.8.1.2) catalyze the hydrolytlc dehalogenation of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids. Four different groups of 2-Haloacid dehalogenases have been found in bacterial cells. The carboxylate group of Asplo of L-2-Haloacid dehalogenase acts as a nucleophile on the a-carbon of L-Zhaloalkanoic acid to form an ester intermdate, which is hydrolyzed to produce the corresponding 2-hydroxyalkanoic acid. In contrast, in the reaction of DL-ZHaloacid dehalogenase (inversion type), a water molecule activated by the enzyme directly attacks the a-carbon of the substrate. D-2-Haloacid dehalogenase shows sequence similarity to DL-ZHaloacid dehalogenase (inversion type), suggesting that the reaction mechanism of D-ZHaloacid dehalogenase is similar to that of DL-?-Haloacid dehalogenase (inversion type). Only the DL-2-Haloacid dehalogenase (retention type) reaction proceeds with retention of the C2-configuration of the substrate, and its reaction mechanism is probably different from those of other 2-Haloacid dehalogenases
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Binding modes of DL-2-Haloacid dehalogenase revealed by crystallography, modeling and isotope effects studies.
Archives of Biochemistry and Biophysics, 2013Co-Authors: Agata Siwek, Tatsuo Kurihara, Nobuyoshi Esaki, Rie Omi, Ken Hirotsu, Keiji Jitsumori, Piotr PanethAbstract:Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-Haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step.
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A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds
Bioscience biotechnology and biochemistry, 2011Co-Authors: Tatsuo KuriharaAbstract: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 ca...
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Mechanism of the Reaction Catalyzed by dl-2-Haloacid Dehalogenase As Determined from Kinetic Isotope Effects†
Biochemistry, 2006Co-Authors: Ewa Papajak, Tatsuo Kurihara, Renata A. Kwiecień, Juliusz Rudzinski, Daria Sicinska, Rafał Kamiński, Łukasz Szatkowski, And Nobuyoshi Esaki, Piotr PanethAbstract:dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-Haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the α-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 ± 0.0001 for (S)-(−)-2-chloropropionate and 1.0082 ± 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on Vmax/KM, 0.78 ± 0.09 for (S)-(−)-2-chloropropionate and 0.90 ± 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Und...
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A new dl-2-Haloacid dehalogenase acting on 2-Haloacid amides: purification, characterization, and mechanism
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Chung Park, Kenji Soda, Tatsuo Kurihara, Tohru Yoshimura, Nobuyoshi EsakiAbstract:Abstract 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.
Jimmy S. H. Tsang - One of the best experts on this subject based on the ideXlab platform.
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Transport of Haloacids across biological membranes.
Biochimica et biophysica acta, 2016Co-Authors: Ka Fai Kong, Jimmy S. H. TsangAbstract:Haloacids are considered to be environmental pollutants, but some of them have also been tested in clinical research. The way that Haloacids are transported across biological membranes is important for both biodegradation and drug delivery purposes. In this review, we will first summarize putative Haloacids transporters and the information about Haloacids transport when studying carboxylates transporters. We will then introduce MCT1 and SLC5A8, which are respective transporter for antitumor agent 3-bromopyruvic acid and dichloroacetic acid, and monochloroacetic acid transporters Deh4p and Dehp2 from a Haloacids-degrading bacterium. Phylogenetic analysis of these Haloacids transporters and other monocarboxylate transporters reveals their evolutionary relationships. Haloacids transporters are not studied to the extent that they deserve compared with their great application potentials, thus future inter-discipline research are desired to better characterize their transport mechanisms for potential applications in both environmental and clinical fields.
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The 228bp upstream non-coding region of Haloacids transporter gene dehp2 has regulated promoter activity.
Gene, 2016Co-Authors: Jimmy S. H. TsangAbstract:Abstract Biodegradation is an effective way to remove environmental pollutants Haloacids, and Haloacids uptake is an important step besides cytoplasmic dehalogenation. Previous study has identified a robust Haloacids transport system in Burkholderia caribensis MBA4 with two homologous genes deh4p and dehp2 as major players. Both genes are inducible by monochloroacetate (MCA), and dehp2 is conserved among the Burkholderia genus with a two component system upstream. Here we show that dehp2 is not in the same operon with the upstream two component system, and fusion with lacZ confirmed the presence of MCA-inducible promoter activity in the 228 bp upstream non-coding region of dehp2. Serial deletion confirmed 112 bp upstream is enough for basic promoter activity, but sequence further upstream is useful for enhanced promoter activity. Electrophoretic mobility shift assay of the 228 bp region showed a retardation complex with stronger hybridization in the induced condition, suggesting a positive regulation pattern. Regulator(s) binding region was found to lie between − 228 to − 113 bp of dehp2. Quantitative real-time PCR showed that the expressions of dehp2 orthologs in three other Burkholderia species were also MCA-inducible, similar as dehp2. The 5′ non-coding regions of these dehp2 orthologs have high sequence similarity with dehp2 promoter, and 100 bp upstream of dehp2 orthologs is especially conserved. Our study identified a promoter of Haloacids transporter gene that is conserved in the Burkholderia genus, which will benefit future exploitation of them for effective biodegradation of Haloacids.
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Draft Genome Sequence of the Haloacid-Degrading Burkholderia
2016Co-Authors: Caribensis Strain Mba, Ka Fai Kong, Yanling Pan, Jimmy S. H. TsangAbstract:Burkholderia caribensisMBA4 was isolated from soil for its ability to utilize 2-Haloacid. An inducible Haloacid operon, encoding a dehalogenase and a permease, is mainly responsible for the biotransformation. Here, we report the draft genome sequence of this strain
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Complete genome sequence and characterization of the Haloacid–degrading Burkholderia caribensis MBA4
Standards in genomic sciences, 2015Co-Authors: Yanling Pan, Ka Fai Kong, Jimmy S. H. TsangAbstract:Burkholderia caribensis MBA4 was isolated from soil for its capability to grow on Haloacids. This bacterium has a genome size of 9,482,704 bp. Here we report the genome sequences and annotation, together with characteristics of the genome. The complete genome sequence consists of three replicons, comprising 9056 protein-coding genes and 80 RNA genes. Genes responsible for dehalogenation and uptake of Haloacids were arranged as an operon. While dehalogenation of haloacetate would produce glycolate, three glycolate operons were identified. Two of these operons contain an upstream glcC regulator gene. It is likely that the expression of one of these operons is responsive to haloacetate. Genes responsible for the metabolism of dehalogenation product of halopropionate were also identified.
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complete genome sequence and characterization of the Haloacid degrading burkholderia caribensis mba4
Standards in Genomic Sciences, 2015Co-Authors: Yanling Pan, Ka Fai Kong, Jimmy S. H. TsangAbstract:Burkholderia caribensis MBA4 was isolated from soil for its capability to grow on Haloacids. This bacterium has a genome size of 9,482,704 bp. Here we report the genome sequences and annotation, together with characteristics of the genome. The complete genome sequence consists of three replicons, comprising 9056 protein-coding genes and 80 RNA genes. Genes responsible for dehalogenation and uptake of Haloacids were arranged as an operon. While dehalogenation of haloacetate would produce glycolate, three glycolate operons were identified. Two of these operons contain an upstream glcC regulator gene. It is likely that the expression of one of these operons is responsive to haloacetate. Genes responsible for the metabolism of dehalogenation product of halopropionate were also identified.
