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.

Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

“…It is therefore not surprising that mutations resulting in modification of this hole result in different effects on different halogenated substrates. This has been demonstrated previously by mutagenesis of the residues in direct contact with the halogen (so-called first-shell residues F151 and F169) (1,21) and also with the second-shell residues F154 (18), I211, and F143 (this study). The constructed mutants, including the sixfold mutant, can be used for further analysis of structure-function relationships of haloalkane dehalogenases.…”

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

“…By analogy to the well-studied mechanism of another haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus (65), the catalytic mechanism of LinB is proposed to involve an interaction of the substrate with the catalytic triad (D108, H272, and E132, i.e., nucleophile-histidine-acid) and a group of halide-stabilizing residues (principally N38 and W109) (18,32,60,130). It was suggested that nucleophilic attack by D108 at the carbon atom of the substrate results in C-Cl bond cleavage by an S N 2 displacement mechanism, resulting in the formation of an acyl-enzyme intermediate.…”

Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of Its Entrance Tunnel

“…Such technologies are already in use (14) or are under development (2, 15, 16). Furthermore, haloalkane dehalogenases has become an important model system for in silico study of molecular principles of enzymatic catalysis (12,(17)(18)(19)(20)(21)(22)(23)(24).Haloalkane dehalogenase LinB (25) is the enzyme isolated from a ␥-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 (26). The LinB enzyme catalyzes conversion of 1,3,4,6-tetrachloro-1,4-cyclohexadiene to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol via 2,4,5-trichloro-2,5-cyclohexadien-1-ol.…”

“…The enzymes cleaves the carbon-halogen bond and replaces a halogen with a hydroxyl group from a water molecule (1). Activity and specificity of haloalkane dehalogenases is not optimal for industrial applications (2), and numerous studies have been conducted to improve their catalytic properties using in vitro techniques (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Engineered enzymes can be used in biotechnology applications, such as detoxification of environmental pollutants and bioorganic synthesis.…”