Elucidating catalytic mechanisms of glycoside hydrolases and transferases by means of ab initio molecular dynamics simulations

• Autores: Javier Iglesias Fernández
• Directores de la Tesis: Josefa Badia Palacín (dir. tes.), Carme Rovira Virgili (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2014
• Idioma: inglés
• Tribunal Calificador de la Tesis: Gabriel Aullón López (presid.), Ramon Hurtado-Guerrero (secret.), Carmen Domene (voc.)
• Materias:
  • Física
    • Química física
      • Teoría cuántica
    • Física del estado sólido
  • Química
    • Bioquímica
      • Enzimología
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • Outline Carbohydrates play a central role in transport and storage of energy and as molecular building blocks. Additionally, glycoconjugates, specifically glycoproteins and glycolipids, are important components of cell surfaces and the extracellular environment that mediate cellular and molecular interactions. Defects in glycosylation are associated with human diseases while the ability of glycans to modulate immune responses leads to them playing a critical role in susceptibility and resistance to pathogens. This huge amount of glycan structures requires the existence of a diverse group of degrading and remodelling enzymes: glycoside hydrolases (GHs) and glycoside transferases (GTs). GHs and GTs are highly specific enzymes responsible of the hydrolysis (GHs) and formation (GTs) of glycosidic bonds in carbohydrates. They are responsible for the modification of polysaccharides and glycoconjugates involved in numerous biological processes such as pathogenesis mechanisms, cell-cell recognition and polysaccharide degradation for biofuel processing. Knowledge of their enzymatic mechanism at a molecular level is crucial to understand how carbohydrates are assembled/degraded in organisms, as well as in developing new drugs. The detailed characterization of the transition state of the chemical reaction in which they participate, for instance, is key for the development of TS-analog inhibitors, which are known to be very efficient. In recent years, our group has investigated the implications of the conformational changes on the substrate during catalysis in several GHs and has related these changes with the conformations that can be sampled by a single sugar unit (e.g. glucose). This was analyzed by adapting sugar puckering coordinates as collective variables in ab initio metadynamics simulations. These studies are having a significant impact not only in the theoretical community but also in biochemistry and biophysics, because of the possibility to predict substrate catalytic itineraries for GHs. In this thesis, we extend these analyses to other sugar molecules to verify the proposed catalytic itineraries and also to GH inhibitors and sugar oxocarbenium ions to gain insights into transition state mimicry. Unlike GHs, known to operate by means of a double displacement mechanism, the reaction mechanism of retaining GTs is controversial. Both a two-step mechanism (by analogy to retaining glycoside hydrolases) and a one-step mechanism have been proposed and studied by means of quantum mechanics / molecular mechanics (QM/MM) simulations. Here, we applied this methodology to elucidate the catalytic mechanism of an engineered glycoside hydrolase and a glycoside transferase, giving support for a front-face single displacement mechanism.