One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the alpha4'-helix-loop-alpha4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.

Department of Inorganic and Physical Chemistry Faculty of Science Palacky University 771 46 Olomouc Czech Republic

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Amadei, A., Linssen, A.B.M., and Berenden, H.J.C. 1993. Essential dynamics of protein. Proteins: Struct. Funct. Gen. 17 412–425. PubMed

Arnold, G.E. and Ornstein, R.L. 1997. Molecular dynamics study of haloalkane dehalogenase; implications for solvent channel access to the active site. In

Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., DiNola, A., and Haak, J.R. 1984. Molecular-dynamics with coupling to an external bath. J. Comp. Phys. 81 3684–3690.

Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham III, T.E., Ross, W.S., Simmerling, C.L., Darden, T.A., Merz, K.M., Stanton, R.V., Cheng, A.L., et al. 1997. AMBER 5.0. University of California, San Francisco.

Copley, S.D. 1998. Microbial dehalogenases: Enzymes recruited to convert xenobiotic substrates. Curr. Opin. Chem. Biol. 2 613–617. PubMed

Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., and Kollman, P.A. 1995. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117 5179–5197.

Damborsky, J. and Koca, J. 1999. Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons. Protein Eng. 12 989–998. PubMed

Damborsky, J., Kuty, M., Nemec, M., and Koca, J. 1997a. A molecular modeling study of the catalytic mechanism of haloalkane dehalogenase: 1. Quantum chemical study of the first reaction step. J. Chem. Inf. Comp. Sci. 37 562–568.

Damborsky, J., Nyandoroh, M.G., Nemec, M., Holoubek, I., Bull, A.T., and Hardman, D.J. 1997b. Some biochemical properties and classification of a range of bacterial haloalkane dehalogenases. Biotech. Appl. Biochem. 26 19–25. PubMed

Damborsky, J., Rorije, E., Jesenska, A., Nagata, Y., Klopman, G., and Peijnenburg, W.J.G.M. 2001. Structure-specificity relationships for haloalkane dehalogenases. Environ. Toxicol. Chem. 20 2681–2689. PubMed

Essmann, U., Perera, L., Berkowitz, M.L., Darden, T.A., Lee, H., and Pedersen, L.G. 1995. A Smooth Particle Mesh Ewald method. J. Chem. Phys. 103 8577–8593.

Goodford, P.J. 1985. A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J. Med. Chem. 28 849–857. PubMed

Hynkova, K., Nagata, Y., Takagi, M., and Damborsky, J. 1999. Identification of the catalytic triad in the haloalkane dehalogenase from PubMed

Janssen, D.B., Pries, F., and Van der Ploeg, J.R. 1994. Genetics and biochemistry of dehalogenating enzymes. Annu. Rev. Microbiol. 48 163–191. PubMed

Janssen, D.B., Scheper, A., Dijkhuizen, L., and Witholt, B. 1985. Degradation of halogenated aliphatic compounds by PubMed PMC

Kabsch, W. and Sander, C. 1983. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22 2577–2637. PubMed

Karplus, M. and McCammon, J.A. 1983. Dynamics of proteins: Elements and functions. Ann. Rev. Biochem. 53 263–300. PubMed

Kennes, C., Pries, F., Krooshof, G.H., Bokma, E., Kingma, J., and Janssen, D.B. 1995. Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity. Eur. J. Biochem. 228 403–407. PubMed

Kmunicek, J., Luengo, S., Gago, F., Ortiz, A.R., Wade, R.C., and Damborsky, J. 2001. Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from PubMed

Koradi, R., Billeter, M., and Wurthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graphics 14 51–55. PubMed

Krooshof, G.H., Floris, R., Tepper, A., and Janssen, D.B. 1999. Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes. Protein Sci. 8 355–360. PubMed PMC

Krooshof, G.H., Kwant, E.M., Damborsky, J., Koca, J., and Janssen, D.B. 1997. Repositioning the catalytic triad acid of haloalkane dehalogenase: Effects on activity and kinetics. Biochemistry 36 9571–9580. PubMed

Laaksonen, L. 2001. gOpenMol 2.1. Espoo, Finland.

Lee, B. and Richards, F.M. 1971. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 55 379–400. PubMed

Lightstone, F.C., Zheng, Y.J., and Bruice, T.C. 1998. Molecular dynamics simulations of ground and transition states for the S(N)2 displacement of Cl

Lightstone, F.C., Zheng, Y.-J., Maulitz, A.H., and Bruice, T.C. 1997. Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an S PubMed PMC

Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., and Damborsky, J. 2000. Crystal structure of the haloalkane dehalogenase from PubMed

Nagata, Y., Miyauchi, K., Damborsky, J., Manova, K., Ansorgova, A., and Takagi, M. 1997. Purification and characterization of haloalkane dehalogenase of a new substrate class from a γ-hexachlorocyclohexane-degrading bacterium, PubMed PMC

Nagata, Y., Miyauchi, K., and Takagi, M. 1999. Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in PubMed

Nardini, M. and Dijsktra, B.W. 1999. α/β Hydrolase fold enzymes: The family keeps growing. Curr. Opin. Struct. Biol. 9 732–737. PubMed

Newman, J., Peat, T.S., Richard, R., Kan, L., Swanson, P.E., Affholter, J.A., Holmes, I.H., Schindler, J.F., Unkefer, C.J., and Terwilliger, T.C. 1999. Haloalkane dehalogenase: Structure of a PubMed

Ollis, D.L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.M., Harel, M., Remington, S.J., Silman, I., Schrag, J., et al. 1992. The α/β hydrolase fold. Protein Eng. 5 197–211. PubMed

Poelarends, G., Zandstra, M., Bosma, T., Kulakov, L.A., Larkin, M.J., Marchesi, J.R., Weightman, A.J., and Janssen, D.B. 2000. Haloalkane-utilizing PubMed PMC

Pries, F., Kingma, J., and Janssen, D.B. 1995a. Activation of an Asp-124–Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine. FEBS Lett. 358 171–174. PubMed

Pries, F., Kingma, J., Krooshof, G.H., Jeronimus-Stratingh, C.M., Bruins, A.P., and Janssen, D.B. 1995b. Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase. J. Biol. Chem. 270 10405–10411. PubMed

Pries, F., Van den Wijngaard, A.J., Bos, R., Pentenga, M., and Janssen, D.B. 1994. The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution. J. Biol. Chem. 269 17490–17494. PubMed

Resat, H. and Mezei, M. 1996. Grand Canonical Ensemble Monte Carlo simulation of the dCpG/proflavine crystal hydrate. Biophys. J. 71 1179–1190. PubMed PMC

Russell, R.B. and Sternberg, M.J.E. 1997. Two new examples of protein structural similarities within the structure-function twilight zone. Protein Eng. 10 333–338. PubMed

Ryckaert, J.P., Ciccotti, G., and Berendsen, H.C. 1977. Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comp. Phys. 23 327–341.

Schanstra, J.P. and Janssen, D.B. 1996. Kinetics of halide release of haloalkane dehalogenase: Evidence for a slow conformational change. Biochemistry 35 5624–5632. PubMed

Schindler, J.F., Naranjo, P.A., Honaberger, D.A., Chang, C.-H., Brainard, J.R., Vanderberg, L.A., and Unkefer, C.J. 1999. Haloalkane dehalogenases: Steady-state kinetics and halide inhibition. Biochemistry 38 5772–5778. PubMed

Schrag, J.D., Winkler, F.K., and Cygler, M. 1992. Pancreatic lipases: Evolutionary intermediates in a positional change of catalytic carboxylates? J. Biol. Chem. 267 4300–4303. PubMed

Slater, J.H., Bull, A.T., and Hardman, D.J. 1995. Microbial dehalogenation. Biodegradation 6 181–189.

van der Spoel, D., van Buuren, A.R., Apol, E., Maulenhoff, P.J., Tieleman, P.D., Sijbers, A.L.T.M., Hess, B., Feenstra, K.A., Lindhal, E., Drunen, R.V., et al. 1999.

Verschueren, K.H.G., Franken, S.M., Rozeboom, H.J., Kalk, K.H., and Dijkstra, B.W. 1993a. Non-covalent binding of the heavy atom compound [Au(CN) PubMed

———. 1993b. Refined X-ray structures of haloalkane dehalogenase at pH 6.2 and pH 8.2 and implications for the reaction mechanism. J. Mol. Biol. 232 856–872. PubMed

Vriend, G. 1990. WHAT IF: A molecular modeling and drug design program. J. Mol. Graphics 8 52–56. PubMed

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