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Author
Sánchez-Izquierdo Besora, Pablo
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Abstract
Rational design via site directed or saturation mutagenesis and directed evolution via random mutagenesis are used as key tools in protein engineering. In both approaches, the sequence diversity is directly generated as point mutation, insertion, or deletion within a single parental gene. Consequently, the improvement in the quality of rationally designed libraries and techniques for sequence space exploration and diversity generation is critical for future advances.
This is why new computational methods has been developed. Computer-Assisted Protein Directed Evolution (CAPDE) methods are within the group of evolution and are based on bioinformatics tools that have been developed for the improvement of the simulation and prediction of the protein design.
In this context, the bioinformatics and molecular modelling unit of the laboratory of Biochemistry at the IQS has developed a computational protocol named BINDSCAN, for assisting in the engineering of enzymes. It is an in-silico tool to predict positions along a protein sequence sensible to the binding of new substrates. To do so it uses different metrics that has been previously created. The objective of this project is to validate BINSCAN for glycoside hydrolases Glycoside hydrolases are a widespread group of enzymes which hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose, hemicellulose, and starch, in anti-bacterial defense strategies, in pathogenesis mechanisms and in normal cellular function. Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.
In this project, BINDSCAN has been validated for two different glycoside hydrolases for which abundant experimental information on mutants is available. The first step is to generate and characterize the enzyme-substrate complex using Autodock software. Once this is accomplished, the next step is to run BINDSCAN. There are 5 principal metrics: Rigid affinity, Lowest energy flexible affinity, Average energy flexible affinity, RMSD and Electrostatic potential (EP) difference. All of them have been tested, characterized, and compared in order to determinate which are the best ones. This will help to understand how BINDSCAN works and also will optimize future runs.
Finally, results have been analyzed qualitatively and quantitatively with a 69% and 100% of success for each enzyme respectively, concluding in that BINDSCAN is a valid CAPDE method for the detection of spots in glycoside hydrolases, especially for those spots that are located at the first shell of the active site.
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