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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/203880
Computational insights into carbohydrate epimerase mechanisms
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[eng] Carbohydrates, as the most abundant biomolecules, play a myriad of roles and functions in biological systems. Unlike the building blocks of proteins, amino acids, whose chemical structure can vary significantly, monosaccharides, the building blocks of carbohydrates, can exhibit small differences in their chemical structure that lead to a very different chemistry. A defining characteristic of monosaccharides is their stereochemistry. Just four or five stereogenic centres in each monosaccharide leads to a vast array of structural possibilities. Given the subtle structural differences between monosaccharides, the enzymes that modify carbohydrates (carbohydrate-active enzymes, CAZymes) must exhibit precise specificity for their substrates. Computational approaches have proven of great value in elucidating the enzymatic mechanisms of CAZymes, particularly in determining the conformation of monosaccharides within the active site during catalysis. Carbohydrate epimerases, a subset of CAZymes, modify the stereogenic centres of carbohydrates. Despite their key roles in biological organisms, such as catalysing the interconversion between glucose and galactose, they remain relatively understudied. The inherent challenge in studying epimerases lies in their need for precise control over the conformation and positioning of the carbohydrate within the active site, given that epimerisation is an equilibrium reaction (i.e., reactant and product have similar energy) and the reactant, intermediates and product are structurally similar. The main applications of carbohydrate epimerases are in biomedicine and biotechnology. Their important roles in humans and other organisms make the research of this group of enzymes essential for biomedical purposes, such as the design of antibiotics. They are also therapeutic targets for the treatment of diseases, such as galactosemia. Furthermore, carbohydrate epimerases can be used to synthesize rare sugars from common ones. While our research group has extensive expertise in CAZymes, it had no experience in carbohydrate epimerases before starting this Thesis. This Thesis summarizes our recent work to uncover some of the innumerable intricacies of carbohydrate epimerases. Following a general introduction and a chapter on the used methods, we elucidate the details of human GDP-L-fucose synthase mechanism. In subsequent chapters, we elucidate the complete mechanism of another biomedically relevant epimerase, UDP-D-glucuronic acid 4- epimerase. - Chapter I: Introduction In this chapter, we introduce the main features of carbohydrates, such as their stereochemistry and conformation, followed by a description of carbohydrate epimerases classification and their mechanisms. The main objectives of this Thesis are detailed at the end of the chapter. - Chapter II: Methods We provide a clear overview of all the computational methodologies employed throughout this Thesis. - Chapter III: Molecular mechanism of regio-selective catalysis in human GDP-L- fucose synthase We uncover the full conformational itinerary of the sugar within the active site human GDP-L-fucose synthase (GFS) throughout its whole catalytic process, and we also elucidate the strategy that the enzyme uses to avoid the premature reduction of the sugar through the precise control of sugar conformation and positioning within the active site. - Chapter IV: Sugar oxidation and rotation in UDP-glucuronic acid C4-epimerisation process We study the oxidation mechanism of the substrate and the elusive mechanism for the rotation of the 4-keto-intermediate within the enzyme active site in Bacillus cereus UDP- D-glucuronic acid C4-epimerase (UGAE). We also discussed how mutations of an important active site residue (Arg185 ) affect catalysis. - Chapter V: Sugar reduction and proton shuttle in UDP-D-glucuronic acid C4- epimerisation process We investigate the mechanism of reduction of UDP-4-keto-hexuronic acid intermediate to UDP-galacturonic acid, which completes the full catalytic mechanism of UDP-D- glucuronic acid C4-epimerase (UGAE).
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ESQUIVIAS BAUTISTA DE LISBONA, Oriol. Computational insights into carbohydrate epimerase mechanisms. [consulta: 10 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/203880]