[en] Carbohydrates are one of the most important biochemical molecules due to their role as energetic resource for non-photosynthetic organisms, cell-cell recognition and adhesion, protection of cell membranes of bacteria and plants and ensure the proper functionality of several enzymes. Glycoproteins are important components of cell surfaces and the extracellular medium where molecular-cell interactions take place. Defects in the activity of glycoproteins are the cause of several human diseases, such as diabetes and lysosomal storage diseases. Glycoside hydrolases (GHs) are one of the most relevant glycoproteins. They catalyse the cleavage of glycosidic bonds of oligosaccharides to generate smaller oligosaccharides or monosaccharides with relevant biological roles. To know the proper way to modulate the activity of these enzymes, it is crucial to understand their molecular mechanisms of action. Sugars are very flexible molecules, most of them formed by 6- membered rings whose conformation changes during the reaction catalysed by GHs. Capturing these conformations, in particular the one at the reaction transition state, is important in inhibitor design. Experiments aimed at characterizing (indirectly) the transition state conformation of GHs include crystallizing the Michaelis complex (MC), which usually is done using thioglycoside derivatives of the natural substrate (i.e. the glycosidic oxygen is substituted by sulfur) or enzyme mutants (e.g. the catalytic acid/base residue mutated to glutamine or asparagine). However, in some cases, the degree of resemblance of the sugar conformation in thioglycosides or enzyme mutants with respect to the natural/wild type counterparts unclear. In last years, our group has demonstrated that the conformational free energy landscape (FEL) of natural sugars (e.g. β-glucose, α,β-mannose and β-xylose) can be used to predict catalytic pathways of GHs. The conformational FEL is explored using Cremer & Pople puckering coordinates as collective variables in the metadynamics method. A natural extension of these studies is to explore the conformational FEL of 7-membered sugar rings (septanosides), which are currently the focus of great interest as potential GH substrates. In this Thesis, we applied Car-Parrinello molecular dynamics methods, within the QM/MM approach, to elucidate the catalytic mechanism of a family 13 retaining GH, (amylosucrase) and a family 125 inverting GH (exo-1,6-α-mannosidase). In both cases, prior structural information was available from enzyme mutants and/or thioderivative substrates. In addition, we have extended previous ring conformational analyses of pyranoses to septanosides in order to assess their potential as new substrates of selected GHs. The Thesis is organised as follows: Chapters I and II focuses on the conformational flexibility of carbohydrates, GH mechanisms and experimental techniques aimed to trap the MC, as well as the methodology used in this Thesis. In Chapter III, we investigate the conformational landscape of α-glucose and the conformational itinerary that an α-glucoside (fructose) follows during catalysis by GH13 amylosucrase (both hydrolysis and polymerization have been investigated). In Chapter IV, we present the results of conformational study of a thioglycoside vs the natural substrate in a GH125 α-mannosidase, including a mechanistic analysis that uncovered the conformational catalytic itinerary for family 125 GHs. In Chapter V, we investigate four MC structures of a promiscuous GH3 β- glucohydrolase, able to cleave several types of glycosidic linkages, for which only structures with thioglycosides are available. The reconstruction of the structure with the natural substrate allowed us to predict the mechanism of action of these enzymes. In Chapter VI, we apply the Cremer & Pople puckering coordinates for 7-membered rings as collective variable for metadynamics on several septanoside molecules (both isolated and "on-enzyme") and assess their potential as substrate/inhibitors of GHs. Finally, Chapter VII contains the main conclusions of this work.

[es] Los carbohidratos son una de las biomoléculas más importantes debido a su papel como fuente de energía para organismos no-fotosintéticos, reconocimiento célula-célula, protección de la membrana celular de bacterias y plantas y por asegurar la buena funcionalidad de algunas enzimas. Las glicoproteínas son componentes esenciales en la superficie de las células y en el medio extracelular. Defectos en la actividad de estas proteínas son la causa de varias enfermedades humanas, como la diabetes o problemas en el lisosoma. Las glicosil hidrolasas (GHs) son una de las glicoproteínas más relevantes. Catalizan la rotura de enlaces glicosídicos de oligosacáridos para generar monosacáridos o cadenas más pequeñas de azúcares con relevancia biológica. Para encontrar una manera adecuada de modular la actividad de estas enzimas, es crucial entender sus mecanismos moleculares. Los azúcares son moléculas muy flexibles, la mayoría formados por anillos de 6 átomos cuya conformación cambia durante la reacción en GHs. Capturar estas conformaciones, en particular la del estado de transición, es clave para diseñar inhibidores. La manera de caracterizar indirectamente la conformación de ese estado de transición es cristalizando el complejo de Michaelis de la GH con un tioderivado del sustrato natural (el oxígeno glicosídico es substituido por azufre) o usando mutantes de la enzima. No obstante, en algunos casos, la semejanza a nivel conformacional del mímico y el sustrato natural no queda suficientemente clara. En los últimos años, nuestro grupo ha demostrado el uso para predecir itinerarios catalíticos de GHs a partir de las superficies de energía libre conformacional de los azúcares naturales (glucosa, manosa y xilosa). Estas superficies se exploran utilizando las coordenadas de empaquetamiento de Cremer y Pople como variables colectivas en el método de metadinámica. Una extensión de estos estudios es el análisis conformacional de azúcares formados por anillos de 7 átomos (septanósidos), que están actualmente en el foco de interés como sustratos de GHs. En la presente Tesis, aplicamos métodos basados en dinámica molecular Car- Parrinello, dentro de la aproximación QM/MM, para estudiar el mecanismo catalítico de las GHs de la familia 13 (amilosucrasa), la familia 125 (exo-1,6-α-manosidasa) y la familia 3 (enzima HvExoI). Además, hemos extendido el estudio conformacional de anillos a los septanósidos para poder predecir su potencial como nuevos sustratos de GHs concretas.