DUGi

Efecte de les mutacions en la variant RA95.5-8F sobre l’activitat retro-aldòlica

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Enzymes, which increase the chemical reactions rate many orders of magnitude, are the most efficient catalysts on Earth. For this reason, and for all their advantageous features, they are becoming very attractive for the industries. However, biocatalyst applied to this field, at this time, is still quite scarce due to some enzymes limitations. These are some barriers that must be overcome. To understand, improve and overcome these barriers, enzyme design is used. This technique can be both experimental and computational, but it always follows the same process. The directed evolution is the most powerful strategy, of the three existing ones, for the enzymes design. This technique has been extensively used, for example for the design of the retro-aldolase 95 (RA95). RA95 promotes the breakdown of carbon-carbon bonds, starting from an aldol and obtaining the corresponding aldehyde and ketone. This reaction is called retro-aldol reaction and specifically promotes the breakdown of the methodol to 6-methoxy-2-naphthaldehyde and acetone. It follows a reaction path of several steps, one of which is the formation of a covalent adduct between the substrate and the catalytic lysine (Schiff base). Several mutations have been introduced into RA95, leading to different variants of the enzyme. The last one, and the most efficient, is RA95.5-8F, which has a catalytic tetrad. This new variant is originated applying some mutations to RA95.5-8 both in the catalytic centre and in the periphery of the enzyme. The aim of this study is to analyse and understand the contribution on catalytic activity of mutated residues that are not in the catalytic centre. Using the “Shortest Path Map” (SPM) tool applied to RA95.5-8, the most relevant residues can be predicted on the basis of the conformational dynamics of the enzyme. Thus, several mutations in RA95.5-8F are reverted back to the original residue found for the RA95.5-8 variant. A molecular dynamics (MD) simulation protocol is followed based on the reaction intermediate formed after the formation of the Schiff base. This allows the rationalization of how the catalytic distance and the "Root Mean Square Deviation" (RMSD) are affected after reverting the selected mutations. In addition, the PyMol program is used for a better visualization of the behaviour of the enzyme during the simulation. The results of the simulations for each of the variants show that the SPM computational tool is a great method to determine which positions of the enzyme are relevant for the retro-aldol activity