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Author
Montsant Fina, Albert
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Abstract
BindScan is a CAPDE (Computer-aided protein directed evolution) tool developed by the Bioinformatics and Molecular Modeling Unit of the Biochemistry Laboratory (IQS). The main purpose for the development of this software is to aid in protein engineering experiments. BindScan is able to predict binding-sensitive positions of non-natural substrates of a protein sequence. This software has been used successfully for the design of synthetic functions of carbohydrate active enzymes (CAZYmes) giving promising results. For example, the design of Lacto-N-biosidase for the synthesis of breast milk oligosaccharides (HMOs), the third most abundant component of milk with many beneficial functions for newborns. In this work, the possibility of using BindScan in other families of enzymes will be explored. This tool is thought to have great potential for predicting spots of a protein sequence that are sensitive to a specific property, which can be very valuable information to take into account when designing a directed evolution experiment.
In this project we want to validate the use of BindScan in other families of enzymes by contrasting published experimental results with those results of simulations obtained using BindScan. For this, a database of enzymes with mutants has been created that contains experimental kinetic data. Of the entries present, methyl parathion hydrolase (MPH), an enzyme capable of degrading pesticides, has been chosen to validate BindScan because it has kinetic parameters defined by a good number of substrates for which native MPH has little catalytic activity. At this point, another objective was incorporated and consisted of using BindScan to modify the substrate specificity of MPH towards three non-natural ligands apart from methyl parathion. Designing an improved MPH with a broader range of substrate specificity is interesting from an environmental point of view since the ligands explored are organophosphates (OPs), toxic components used in many insecticides and that cause diseases. The modes of binding between MPH and four different substrates were predicted with Autodock. BindScan was then used to screen for MPH mutant libraries and to analyze improvements in binding affinity. BindScan has been able to predict hot spots and cold spots in the MPH sequence for coupling with four substrates. These results have been compared with the literature, showing a certain correlation between the BindScan affinity result and the Michaelis constant KM, which seems reasonable since this kinetic parameter provides information on the affinity in an enzyme-substrate interaction. The results obtained from the simulations are not inconsistent with the literature but it is believed that more experimental data from more mutants were needed to establish well-defined and reliable correlations that would validate BindScan.
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