1National Centre for Biomolecular Research, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic and 2Graduate School of Life Sciences, Tohoku University, Katahira, Sendai 980-8577, Japan.
Haloalkane dehalogenases are bacterial enzymes catalyzing the hydrolytic cleavage of the carbon-halogen bond of various halogenated aliphatic hydrocarbons present in the environment as dangerous pollutants. This catalytic process results in the formation of an alcohol, halide and proton as the reaction products. Catalytic triad, oxyanion hole and halide-stabilizing residues were previously found to be the three main structural features essential for the catalytic performance in these enzymes . Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The difference in halide-stabilizing residues between two studied haloalkane dehalogenases with known structure was tested in present study using quantum mechanical calculations and site-directed mutagenesis.
Nucleophilic substitution (SN2) as the first reaction step of dehalogenation process was modeled and the level of electrostatic stabilization of the transition state structure and released halide ion provided by each of the active site residues was calculated. Studied systems were the enzymes DhlA from the soil bacterium Xanthobacter autotrophicus GJ10 and LinB from Sphingomonas paucimobilis UT26 and 1-chlorobutane substrate docked into the active site of the protein. Our in-house program TRITON [2,3] interfacing semi-empirical quantum mechanical package MOPAC  and homology modeling package MODELLER  was used for all calculations. Presented results showed that some of the studied residues can be assigned as primary (essential) and some as secondary (less important) halide-stabilizing residues. Consecutively both theoretical and experimental site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary and secondary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr and Phe169Leu mutants of LinB were theoretically modeled and simultaneously experimentally constructed, purified and kinetically characterized.
Based on the results the following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA, and Asn38 and Trp109 of LinB. All these residues stabilize the halide ion released from the substrate molecule by a hydrogen bond and their substitution significantly modified catalytic properties of mutated enzymes. Phe172, Pro223 and Val226 of DhlA and Trp207, Pro208 and Ile211 of LinB residues were classified as the secondary halide-stabilizing residues with no significant effect on the catalysis. The good qualitative agreement between modeled stabilization effect and catalytic activity for studied mutants of LinB was observed  confirming the applicability of TRITON for similar type of studies. Program TRITON is currently available for Irix, Linux and NetBSD operating systems and is provided free of charge for academic users. For more information and program download see the web page http://ncbr.chemi.muni.cz/triton/triton.html.
1. Verschueren, K. H. G., Seljee, F., Rozeboom, H. J., Kalk, K. H., and Dijkstra, B. W., Nature 363 (1993) 693-698.
2. Damborský, J., Prokop, M., and Koča, J., Trends in Biochemical Sciences 26 (2001) 71-73.
3. Prokop, M., Damborský, J., and Koča, J., Bioinformatics 16 (2000) 845-846.
4. Stewart, J. J. P. (1990) Journal of Computer-Aided Molecular Design 4, 1-45.
5. Sali, A., Molecular Medicine Today 1 (1995) 270-277.
6. Boháč, M., Nagata, Y., Prokop, Z., Prokop, M., Monincová, M., Koča, J., Tsuda, M., and Damborský, J., Biochemistry 41 (2002) 14272-14280.