Structure-activity investigation on laccases by computational and site directed mutagenesis studies
Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/404452 |
| Acceso en línea: | http://hdl.handle.net/10803/404452 https://dx.doi.org/10.5821/dissertation-2117-106481 |
| Access Level: | acceso abierto |
| Palabra clave: | Àrees temàtiques de la UPC::Enginyeria agroalimentària 577 663/664 |
| Sumario: | Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 site is shuttling electrons from the substrate to the trinuclear copper cluster. During the catalytic cycle of laccase, four electrons are removed from four substrate molecules, which are finally transferred to reduce oxygen to two water molecules .Comparison of the kinetic parameters using several laccases and several substrates reveals that the reaction rate of laccase correlates with the redox potential difference between the T1 copper and the substrate. In recent years, the demonstrated potential of laccases in a range of applications has motivated the progress of laccase engineering efforts. Computational simulations can reveal targets for protein engineering to be explored by site-directed mutagenesis (or semi-rational approaches). In this work we used computational methods for studying interaction of different substrates with laccases and structural activity of the enzyme. The goal of the present study was to characterize the laccase binding pocket of fungal and bacterial laccases in order to establish their common pharmacophoríc characteristics. For this purpose, we first performed molecular docking studies to identify those residues involved in the interaction with diverse substrates. Our results indicate that bacterial laccase {1UVW) has less hydrophobic and aromatic residues in the activity site in comparison to other fugal structures of this study, as a result, find a pose that interacts with residues needs more energy. Subsequently, we evaluated the effect of protonation state of a conserved residue in fungal laccase, Asp/Glu, through molecular dynamics simulation. In a subsequent step, we applied QMMM-2QM-MD approach for one of the fungal laccase structure (3FU8) for calculating redox potential value. The result indicates that the difference in redox potentials changes from 7-17 to 74-92 kJ/mol if the redox state of T1Cu and DMP in the other subunit change and we correctly predict that CuT1ox/DMPred state is more stable than the CuT1red/DMPox state. After the insight gathered from computational studies we started site directed mutagenesis studies on two residues of the binding pocket in order to find their effect on the redox potential value. We made a combinatorial library for position 192 and 296 in MtlL T2. The clone contained A192P and L296W (3H12) mutation and clone contained A192P and 296L {19G8) showed activity with violuríc acid 1.23 and 1.33 fold higher than parental type, respectively. Moreover, the clone contained A192R and L296W (15H11) and clone with mutation A192R and L296L {5B4) showed higher activity with molybdenum compound in comparison to parental type. After experimental characterization of the 19G8 and 5B4 mutants, we studied the structural changes produced in the binding pocket. For this purpose we generated a three-dimensional structure of the two mutants using M.albomices laccase as template by homology modelling. Whereas the former mutant exhibits a similar binding pocket to the template, the latter appears to be smaller. In any case, subsequent docking studies did not show any differential behaviour and ligands could bind to both binding pockets in a similar way. Finally, we calculated the redox potential of the mutant A296L MaL that is similar to the former mutant, yielding a value of 167 kJ/mol. This is higher than the value obtained for MalL supporting the effect of this mutation on the redox potential. |
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