Rational enzyme engineering of heme peroxidases through biophysical and biochemical modeling
Enzymes are proteins that catalyze biochemical reactions and their use report multiple advantages, as they can be very selective, low polluting (biodegradable), cheap and allow working in mild conditions compared with traditional non enzymatic processes. Despite their enormous benefits, their applic...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Recursos: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/399735 |
| Acesso em linha: | http://hdl.handle.net/10803/399735 |
| Access Level: | acceso abierto |
| Palavra-chave: | Enzims Enzimas Enzymes Peroxidasa Peroxidase Ciències Experimentals i Matemàtiques 544 |
| Resumo: | Enzymes are proteins that catalyze biochemical reactions and their use report multiple advantages, as they can be very selective, low polluting (biodegradable), cheap and allow working in mild conditions compared with traditional non enzymatic processes. Despite their enormous benefits, their applications at the industrial level are still limited, mainly due to low productivity, low substrate tolerance (too specifics) and poor resistance to the industrial conditions, and for this reason, developing enhanced enzymes by means of enzyme engineering is a central research field nowadays. Notably, the application of computational chemistry in the field of enzyme engineering is increasing due to improvements in hardware and software. Moreover, this process is fast and low-priced and therefore, profitable for the application to the real problems that face industry. Therefore, motivated by this progress, the main goal of this thesis is the development of computational strategies that allow designing and evaluating modifications in enzymes, also aiming to obtain results quickly and inexpensively. This purpose was reached by the combination of different in silico methodologies that were further supported by experimental data in an interactive feedback process. As a result of this thesis, the enzymatic process in heme peroxidases was first satisfactorily described by dividing the process into two steps (from the ligand diffusion to the chemical reaction), using a combination of different computational techniques. The first step, which involves the protein/ligand recognition, was characterized with different molecular mechanics based techniques (MD, Docking and MC-PELE). On the other hand, the chemical reaction (including bond formation and electron transfer) was reproduced using QM based methods by means of energy calculation, spin density characterization, e-coupling calculations and QM/MM e-pathways descriptions. Following this procedure, the oxidation of veratryl alcohol by the enzyme lignin peroxidase was also characterized. Moreover, regarding the e-coupling calculations, a server to compute this vale faster and easy was developed. In the second part of the thesis, the results demonstrated that our protocol could reliably describe and predict enzymatic functions, not only in native enzymes but also in mutated ones, which results were in agreement with experimental data. For example, the structural implications over the reactivity in manganese peroxidase and its engineered variant obtained by cutting the last terminal residues were identified and characterized by the combination of Monte Carlo simulations (PELE) and electronic coupling calculations. The pH resistance in the mutant 2-1B (which was obtained experimentally by random directed evolution) in contrast with the wild type versatile peroxidase, were also rationalized by molecular dynamics, where the residues in the heme environment presented different conformation due to the mutations introduced, resulting in different pH resistance. Interestingly, the last part of the thesis was centered of engineering heme peroxidases. We engineered a peroxidase from in silico predictions to elucidate the long range electron transfer processes involved in the oxidation of the substrate veratryl alcohol by the enzyme versatile peroxidase. In this work we identify the key residues involved in the process, with further applications in engineering enhanced enzymes. Moreover, an enhanced manganese peroxidase mutant from a complete computational study was designed. First, the ligand diffusion study allowed finding the key aminoacids in the substrate/enzyme recognition and binding. Then, the chemical reaction in terms of the oxidation probability and kinetic constant for the proposed mutant were estimated, and the results were in agreement with experimental data. Therefore, the work of this thesis probed that computational biophysics and biochemistry are promising and valuable tools for enzyme engineering. In particular, in the field of rational design of heme peroxidases, they provide relevant information about the enzymatic mechanism and allow designing new enzymes, as well as checking their improvement/worsening, in an efficient way. |
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