Publication details
Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis
| Authors | |
|---|---|
| Year of publication | 2002 |
| Type | Article in Periodical |
| Magazine / Source | Biochemistry |
| MU Faculty or unit | |
| Citation | |
| Web | http://ncbr.chemi.muni.cz/~jiri/ABSTRACTS/biochem02b.html |
| Field | Biochemistry |
| Keywords | QUANTUM MECHANICS; HALIDE; MUTANT; PROTEIN ENGINEERING; STABILISATION |
| Description | Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds resulting in the formation of an alcohol, a halide and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of SN2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr and Phe169Leu mutants of LinB were constructed, purified and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223 and Val226 of DhlA; Trp207, Pro208 and Ile211 of LinB; and Phe205, Pro206 and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates. |
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