Structure–specificity relationships for haloalkane dehalogenases

Damborský, Jiřı́; Rorije, Emiel; Jesenská, Andrea; Nagata, Yasunobu; Klopman, Gilles; Peijnenburg, Willie J.G.M.

doi:10.1002/etc.5620201205

Cited by 56 publications

(38 citation statements)

References 39 publications

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“…Similarly, the architecture of the cap domain of DhlA is different from that in LinB and DhaA (Fig. 7), and the substrate specificity of DhlA is significantly different from the specificity of LinB and DhaA (Damborsky et al 1997b;Damborsky et al 2001). The essential motions of DhlA are spread over the entire cap domain.…”

Section: Discussionmentioning

confidence: 97%

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

Otyepka¹

2002

Protein Science

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One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the ␣4Ј-helix-loop-␣4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.

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Section: Discussionmentioning

confidence: 97%

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

Otyepka¹

2002

Protein Science

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“…Activity assays. Haloalkane dehalogenase activities in crude extracts were determined in triplicate by a microtiter plate colorimetric assay by using the reagents of Iwasaki et al (11) as described previously by Damborsky et al (8). A precise activity assay was conducted by using gas chromatography to determine both substrate and product concentrations in the reaction mixture as described by Jesenská et al (14).…”

Section: Methodsmentioning

confidence: 99%

“…The substrate specificity of crude extracts prepared from E. coli GI724 (dhmA) was essentially the same as the substrate specificity of crude extracts prepared from M. avium MU1 ( Table 2). The substrate specificity of DhmA is unlike the substrate specificities of LinB, DhaA, and DhlA dehalogenases (8). A biotechnologically interesting observation is the good activity of this protein with the priority pollutant 1,2-dichloroethane (32), which may be related to higher sequence identity between DhmA and DhlA than between DhmA and LinB or DhaA.…”

Section: Vol 68 2002mentioning

confidence: 99%

Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA

Jesenská¹,

Bartoš

Czerneková

et al. 2002

Appl Environ Microbiol

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Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G؉C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.Haloalkane dehalogenases catalyze hydrolytic cleavage of carbon-halogen bonds in halogenated aliphatic compounds, leading to the formation of primary alcohols, halide ions, and protons. These enzymes are potentially useful for cleaning up contaminated subsurfaces (32) and for processing by-products of chemical syntheses (33). Haloalkane dehalogenases can serve as a model system for studies of the evolution and distribution of degradation enzymes in the environment since many of these enzymes have already been isolated from different bacterial species originating from geographically distinct areas (26). Haloalkane dehalogenases have primarily been isolated from bacteria colonizing environments contaminated by halogenated substances (12,16,22,(27)(28)(29)(30)40). Only recently have the hydrolytic dehalogenation activities of several species of the genus Mycobacterium isolated from clinical material been reported (14). Motivation for the search for haloalkane dehalogenases in clinical samples of mycobacteria came from the identification of dehalogenase-like genes in the genome of Mycobacterium tuberculosis...

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“…The major catalytic domain of LinB belongs to the large and well-characterized ␣/␤-hydrolase fold superfamily of proteins, which characteristically carry out two-step hydrolytic reactions driven by a nucleophile (Asp) that is part of a catalytic triad and using an oxanion hole to stabilize the intermediate (69,114,117). LinB has been the subject of several crystallographic (92,123,166) and computational studies (32,33,69,122,130).…”

Section: Linb Haloalkane Dehalogenase Biochemistrymentioning

confidence: 99%

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

Lal

Pandey

Sharma

et al. 2010

Microbiol Mol Biol Rev

349

338

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SUMMARY Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

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Structure–specificity relationships for haloalkane dehalogenases

Cited by 56 publications

References 39 publications

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

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