Esterases computational study

In our project, we are focusing on two different esterases called LAE6 and LAE5. LAE6 is very promiscuous since it presents activity towards a wide range of different substrates, whereas LAE5 presents activity towards few substrates. This promiscuity can be due to the location of the binding cavity: in LAE6 the binding cavity is located buried inside the protein, so the substrates can be retained longer, correlating with a higher reactive probability . LAE5 binding cavity is situated on the surface of the protein, in direct contact with the solvent, which, in our working hypothesis, is the main reason for its much less promiscuity. We are using several computational techniques to extract important descriptors in order to construct a mathematical model to predict the activity of these two enzymes towards different substrates

THE MOLT ISSUE: WHERE DO WE GO FROM NOW?

No abstract

Las soluciones cristianas al problema social

Sesión del día 7 de febrero de 1921Copia Digital : Real Academia de Jurisprudencia y Legislación, 201

THE MOLT ISSUE: WHERE DO WE GO FROM NOW?

No abstract

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Computer-aided laccase engineering: toward biological oxidation of arylamines

Oxidation of arylamines, such as aniline, is of high industrial interest and laccases have been proposed as biocata-lysts to replace harsh chemical oxidants. However, the reaction is hampered by the redox potential of the substrate at acid pH and enzyme engineering is required to improve the oxidation. In this work, instead of trying to improve the redox potential of the en-zyme, we aim towards the (transient) substrate’s one and propose this as a more reliable strategy. We have successfully combined a computational approach with experimental validation to rationally design an improved biocatalyst. The in silico protocol combines classical and quantum mechanics to deliver atomic and electronic level detail on the two main processes involved: substrate binding and electron transfer. After mutant expression and comparison to the parent type, kinetic results show that the protocol accurately predicts aniline’s improved oxidation (2-fold kcat increase) in the engineered variant for biocatalyzed polyaniline production.This study was supported by the INDOX (KBBE-2013-7-613549) EU-project, and the NOESIS (BI0201456388-R) and OxiDesign (CTQ2013-48287-R) Spanish project. GS thanks an FPI grant of the Spanish Ministry of Competitiveness.Peer ReviewedPostprint (author's final draft

Rational Engineering of Multiple Active Sites in an Ester Hydrolase

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes