Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity.

University of Groningen REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCES HALIDE BINDING AND CATALYTIC ACTIVITY

Crystallographic and fluorescence studies of the interaction of haloalkane dehalogenase with halide ions. Studies with halide compounds reveal a halide binding site in the active site.

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 catalyzes the conversion of 1,2-dichloroethane to 2-chloroethanol and chloride without use of oxygen or cofactors. The active site is situated in an internal cavity, which is accessible from the solvent, even in the crystal. Crystal structures of the dehalogenase enzyme complexed with iodoacetamide, chloroacetamide, iodide, and chlori...

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase.

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state k(cat) with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Cl alpha) from 1,2-dichlo...

The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution.

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known h...

Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes.

Haloalkane dehalogenase (DhlA) hydrolyzes short-chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the thre...