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 [1]. 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 [4] and homology modeling package MODELLER [5] 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 [6] 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.
References:
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.