Theoretical and experimental study of the reaction mechanism of several lipoxygenases and hydrolases: governing their product pattern by mutagenesis

Abstract

Lipoxygenases are a family of enzymes involved in the immune system, which are able to oxidise in a stereo- and regioselective way a family of compounds called polyunsaturated fatty acids. The product of these biochemical reactions is a series of biosignalling compounds involved in inflammation and its resolution. Along this Thesis, four different lipoxygenases have been studied focusing on the differences between two enzymes codified in the same gene of two distinct mammalian species, on the effect of the application of mutations in enzyme's key regions for the modification of the stereo- and regioselectivities of the enzyme, and on the biocatalytic mechanism that drives the transformations of the fatty acids into biosignalling molecules. Moreover, the role of the two proteins closely related with lipoxygenases has been studied. On the one hand, Five-Lipoxygenase-Activating protein, necessary for the proper function of the Lipoxigenase-5, has been studied in a nuclear membrane's environment. On the other hand, the biocatalytic mechanism of the Leukotriene A4 Hydrolase has been studied. This enzyme participates in the synthesis of the Leukotriene B4, a key compound in the inflammation stage. Furthermore, mutants of Leukotriene A4 Hydrolase have been designed focusing on its enzymatic cavity to allow the reactivity of derivatives of the docosahexaenoic acid in order to make it possible to be used in an industrial synthesis of Maresin-1, one of the most potent biosignalling molecules for the resolution of the inflammation, so it can be applicable as a drug for patients with chronic inflammatory diseases such as COVID-19, ALS or MS, among others._x000D_ _x000D_ These studies have been carried out from two different perspectives: the experimental and the computational. On the one hand, some of the studied enzymes have been expressed and incubated with fatty acids, and the resulting products have been analysed to understand the stereo- and regioselectivity of the enzymes. On the other hand, several methods from the Computational Chemistry field such as molecular Docking, molecular dynamics and QM/MM calculations have been applied to explore different aspects of the enzymes, from the protein-substrate interaction to the biocatalytic mechanism at the quantum level of theory.

Date of Award	20 Dec 2023
Original language	English
Supervisor	M. Dels Angels Gonzalez Lafont (Director) & Jose Mª Lluch Lopez (Director)