Comparative binding energy (COMBINE) analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10
| Autoři | |
|---|---|
| Rok publikování | 2001 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Biochemistry |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | http://www.ncbr.chemi.muni.cz/~jiri/ABSTRACTS/biochem01.html |
| Obor | Biochemie |
| Popis | Comparative binding energy (COMBINE) analysis was conducted for eighteen substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10: 1-chlorobutane; 1-chlorohexane; dichloromethane; 1,2-dichloroethane; 1,2-dichloropropane; 2-chloroethanol; epichlorohydrine; 2-chloroacetonitrile, 2-chloroacetamide and their brominated analogs. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91 % (73 % cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93 % (74 % cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223 and Leu263. These residues are suitable targets for modification by site-directed mutagenesis. |
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