title: Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis.
abstract: 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 S(N)2 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.
authors.affiliation: National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Bohác
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
authors.forename: Martin
authors.initials: M
authors.lastname: Prokop
authors.forename: Marta
authors.initials: M
authors.lastname: Monincová
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Koca
authors.forename: Jirí
authors.initials: J
authors.lastname: Damborský
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000838:Anions / Q000737:chemistry; D001216:Asparagine / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006219:Halogens / Q000737:chemistry*; D006639:Histidine / Q000235:genetics; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics*; D007532:Isoleucine / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008432:Mathematical Computing; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011392:Proline / Q000235:genetics; D011789:Quantum Theory; D055672:Static Electricity; D014364:Tryptophan / Q000235:genetics; D014633:Valine / Q000235:genetics
chemical_list: D000596:Amino Acids; D000838:Anions; D006219:Halogens; D007532:Isoleucine; D018698:Glutamic Acid; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D014364:Tryptophan; D011392:Proline; D006867:Hydrolases; C113022:haloalcohol dehalogenase; C046137:haloalkane dehalogenase; D007930:Leucine; D014633:Valine