Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a <i>Rhodococcus</i> Haloalkane Dehalogenase

Bosma, Tjibbe; Pikkemaat, M.G.; Kingma, Jaap; Hoogesteger, Jaime; Janssen, Dick B.

doi:10.1021/bi026907m

Cited by 43 publications

(36 citation statements)

References 37 publications

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“…e Determined at pH 8.2 and 30°C (31). f Determined at pH 9.4 and 30°C (14). g Determined at pH 8.6 and 37°C (this study).…”

Section: Discussionmentioning

confidence: 75%

“…The main and very important difference in kinetic mechanism is in the rate-limiting step. Whereas the halide release is the predominant rate-limiting step of DhlA (8) and liberation of alcohol is the rate-limiting step of DhaA (14) under steady-state conditions, the release of both halide and alcohol was found to be fast in the catalytic cycle of LinB. The conclusion that there are different rate-limiting steps for three enzymes from the same protein family sharing the same chemical mechanism demonstrates that extrapolation of this important catalytic property of one enzyme to another can be misleading, even for evolutionary closely related proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Three haloalkane dehalogenases representing these different classes have been isolated and characterized in detail so far: the haloalkane dehalogenase DhlA (12), the haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 (11) and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 (13). The kinetic mechanism has been solved for DhlA (9) and DhaA (14). Comparison of the kinetic mechanism of DhlA and DhaA shows that the overall scheme is similar.…”

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confidence: 99%

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Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Prokop

Monincová

Chaloupková

et al. 2003

Journal of Biological Chemistry

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Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.

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“…e Determined at pH 8.2 and 30°C (31). f Determined at pH 9.4 and 30°C (14). g Determined at pH 8.6 and 37°C (this study).…”

Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Prokop

Monincová

Chaloupková

et al. 2003

Journal of Biological Chemistry

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“…Steady-state kinetic constants were determined for DmmA short (Table II), which revealed that these parameters are comparable to other HLDs. 15,27,29 DhlA has threefold greater catalytic efficiency than DmmA for 1,3-dibromopropane (fourfold lower k cat , 12-fold lower K m ), 27 whereas…”

Section: Hld Activitymentioning

confidence: 99%

Structure and activity of DmmA, a marine haloalkane dehalogenase

Gehret¹,

Gu²,

Geders³

et al. 2012

Protein Science

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DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K m 5 0.24 6 0.05 mM, k cat 5 2.4 6 0.1 s 21). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.

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“…The differences in the substrate specificities of these three haloalkane dehalogenases can be accounted for on the basis of their three-dimensional structures (11). Comparison of the kinetic mechanisms of DhlA, DhaA, and LinB showed that the overall reaction mechanisms are similar but that the rate-limiting steps differ, i.e., halide release in the case of DhlA (44), liberation of an alcohol in the case of DhaA (4), and hydrolysis of an alkyl-enzyme intermediate in the case of LinB (41). Partial improvement in the catalytic properties and modification of the substrate specificities of haloalkane dehalogenases by rational design (5,34) and directed evolution approaches (3,40) have recently been reported.…”

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confidence: 99%

Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities

Sato

Monincová

Chaloupková

et al. 2005

Appl Environ Microbiol

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Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards ␤-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more ␣-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic compounds that occur as soil pollutants (1,17,38). Haloalkane dehalogenases catalyze the hydrolytic cleavage of the carbon-halogen bond(s) and produce the corresponding alcohols, halide ions, and protons. These enzymes belong to the ␣/␤-hydrolase superfamily (39).Haloalkane dehalogenases are attractive targets for proteinengineering studies aimed at improving catalytic efficiency and at broadening the range of substrate specificity for important environmental pollutants. To date, the three-dimensional structures of three haloalkane dehalogenases have been determined by protein crystallography: DhlA from Xanthobacter autotrophicus GJ10 (48), DhaA from Rhodococcus sp. (25, 35), and LinB from Sphingomonas paucimobilis UT26 (29). The differences in the substrate specificities of these three haloalkane dehalogenases can be accounted for on the basis of their three-dimensional structures (11). Comparison of the kinetic mechanisms of DhlA, DhaA, and LinB showed that the overall reaction mechanisms are similar but that the rate-limiting steps differ, i.e., halide release in the case of DhlA (44), liberation of an alcohol in the case of DhaA (4), and hydrolysis of an alkyl-enzyme intermediate in the case of LinB (41). Partial improvement in the catalytic properties and modification of the substrate specificities of haloalkane dehalogenases by rational design (5, 34) and directed evolution approaches (3, 40) have recently been reported. However, it remains difficult to construct mutant enzymes with entirely new capabilities using only protein-engineering techniques, and therefore, the isolation of new family members is still desirable.For quite some time, haloalkane ...

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Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

Abstract: . (2003). Steady-state and pre-steadystate kinetic analysis of halopropane conversion by a Rhodococcus haloalkane dehalogenase. Biochemistry, 42(26), 8047-8053.

Cited by 43 publications

References 37 publications

Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Structure and activity of DmmA, a marine haloalkane dehalogenase

Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities

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