Purification and Properties of 2-Halo Acid Dehalogenase from<i>Pseudomonas putida</i>

Motosugi, Kenzo; Esaki, Nobuyoshi; Soda, Kenji

doi:10.1080/00021369.1982.10865158

Cited by 4 publications

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“…L-DEX YL has a catalytic nucleophile, Asp-10 (13). 2 The ester intermediate is formed from Asp-10 and the substrate in the enzyme reaction, and hydrolyzed by the solvent water. This mechanism is quite similar to that of HAL, which belongs to the ␣/␤ hydrolase fold family.…”

Section: Possible Roles Of the Active Site Residues Of L-dex Yl-mentioning

confidence: 99%

“…Several bacterial strains have been found to take up the compounds as sole carbon and energy sources and to degrade them using enzymes called dehalogenases. Bacterial 2-haloacid dehalogenases (EC 3.8.1.2) isolated so far from Pseudomonas (1)(2)(3)(4)(5)(6) and Xanthobacter (7) spp. catalyze the hydrolytic cleavage of carbon-halogen bonds of 2-haloalkanoic acids (Reaction 1):…”

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confidence: 99%

“…The catalytic mechanism of L-DEX YL has been studied by comprehensive site-directed mutagenesis (12), 18 O incorporation (13), and chemical modification with hydroxylamine. 2 These studies revealed that Asp-10, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180 are important for the enzymatic activity, and that Asp-10 plays an essential role as a nucleophile in the enzymatic reaction. Consequently, the following mechanism is proposed for the hydrolytic reaction.…”

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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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153

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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Section: Possible Roles Of the Active Site Residues Of L-dex Yl-mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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153

114

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“…There have been very few reports available on degradation of halogenated aliphatic compounds especially 3chloropropionate (3CP). Moreover, many studies have focused on biodegradation of 2-haloacid or α-chloro substituted alkanoates, such as 2,2-dichloropropionic acid (2,2DCP) and D,L2-chloropropionic acid (D,L2CP) (Motosugi et al, 1982;Barth et al, 1992;Thasif et al, 2009;Wong and Huyop, 2011), whereas the degradation of β-chloro substituited alkanoates (3CP) has not been well documented and there are very few reports to date on degradation of this compound by bacteria and no report so far from fungal (Mesri et al, 2009;Yusn and Huyop, 2009). Therefore, current study reports on the identification of fungal species and degradation of 3CP by two fungal isolates.…”

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confidence: 99%

Molecular identification and biodegradation of 3-chloropropionic acid (3CP) by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

Parvizpour¹,

Hamid²,

Huyop³

2013

MJM

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Aims: This study was carried out to further characterize fungal species that could degrade 3-chloropropionic acid (3CP) as sole source of carbon and energy. Methodology and Results: Both fungi were able to grow on 3CP after 10 days on solid minimal media. Based on sequencing of its segment of 18S rRNA these isolates were identified as Mucor sp. SP1 and Trichoderma sp. SP2. The isolated strains were not able to grow on media plates containing 10 mM of 2,2-dichloropropionate (2,2DCP) as sole source of carbon. 3CP degradation was observed in liquid minimal medium containing 10 mM 3CP after 18 days culture period. The chloride ion released was detected in both growth medium containing Mucor sp. SP1 and Trichoderma sp. SP2. At least 80% of 10 mM 3CP was utilized in the growth medium. Conclusion, significance and impact of study: Dehalogenase enzyme that can degrade α-chloro-substituted haloalkanoic acids for example 2,2DCP is well studied up to protein crystallization. Very few reports on the degradation of β-chloro-substituted haloalkanoic acids such as 3CP and none from fungi. This study is considered important because it can be compared to that of well-documented α-chloro-substituted haloalkanoic acids degradation. This is the first study to indicate fungal growth on 3CP as sole carbon and energy sources.

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“…1. 2), a member of the superfamily of HAD hydrolases that includes phosphatases, epoxide hydrolases, and L-2-haloacid dehalogenases (9), catalyzes the hydrolytic dehalogenation of L-2-haloalkanoates to produce the corresponding D-2-hydroxyalkanoates in a mechanism that is very reminiscent of the ATP hydrolysis catalyzed by ATPases (10,11).…”

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Lys⁶⁹¹and Asp⁷¹⁴of the Na⁺/K⁺-ATPase α Subunit Are Essential for Phosphorylation, Dephosphorylation, and Enzyme Turnover

Scheiner‐Bobis

2004

Biochemistry

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P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.

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Purification and Properties of 2-Halo Acid Dehalogenase fromPseudomonas putida

Cited by 4 publications

References 1 publication

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

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

Molecular identification and biodegradation of 3-chloropropionic acid (3CP) by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

Lys⁶⁹¹and Asp⁷¹⁴of the Na⁺/K⁺-ATPase α Subunit Are Essential for Phosphorylation, Dephosphorylation, and Enzyme Turnover

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Purification and Properties of 2-Halo Acid Dehalogenase fromPseudomonas putida

Cited by 4 publications

References 1 publication

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

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

Molecular identification and biodegradation of 3-chloropropionic acid (3CP) by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

Lys691and Asp714of the Na+/K+-ATPase α Subunit Are Essential for Phosphorylation, Dephosphorylation, and Enzyme Turnover

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Lys⁶⁹¹and Asp⁷¹⁴of the Na⁺/K⁺-ATPase α Subunit Are Essential for Phosphorylation, Dephosphorylation, and Enzyme Turnover