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.

National Centre for Biomolecular Research Masaryk University Kotlarska 2 611 37 Brno Czech Republic

Citace poskytuje Crossref.org

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards β-hexachlorocyclohexane

Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate

Amperometric Sensor for Detection of Chloride Ions