Dissertação para obtenção do Grau de Doutor em
Bioquímica – Ramo Bioquímica EstruturalThe microbial plant cell wall degradation is one of the most important processes in the global turnover of atmospheric carbon dioxide. The work presented in this thesis addressed the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens, essential to the process of cellulose degradation, and aimed to study some of the components involved in their architecture (cohesins and dockerins) and efficiency (Carbohydrate-Binding Modules - CBMs). For this I used a combination of Nuclear Magnetic Resonance (NMR), X-ray crystallography and computer modeling techniques. My objective was to help rationalize the molecular determinants of specificity of CBMs, including the CtCBMs of families 11, 30 and 44, and the mechanisms of molecular recognition between cohesins and dockerins. In Chapter I, I present a general introduction to the theme of degradation of plant cell walls, with special attention to the cellulosome and its components. In Chapter II, I discuss the structural characteristics of the CtCBM11 based on the structures obtained by NMR at 25 and 50 °C and the structure obtained by crystallography. I found that although similar, the structures show some differences, particularly regarding the binding cleft area, which explains the negative results obtained by co-crystallization. In Chapter III and IV I study the molecular determinants of specificity in modules CtCBM11, 30 and 44, based on NMR and computer modeling data. I found that the atoms of the cellooligosaccharides most important for binding are the ones at positions 2 and 6 of the central units of the ligands. Moreover, I characterized the mechanisms responsible for selection and binding of these modules to various substrates. I established that binding occurs by a mechanism for conformational selection, where the topology of the residues of the protein, the conformation of the ligand and the number of glucose units, play a fundamental role. Chapters V and VI reveal the determination of the 3D structure of the cohesin-module X-dockerin complex of C. thermocellum and the cohesin-dockerin complex of B. cellulosolvens, respectively. Both complexes belong to the type II and their analysis allowed obtaining important information about the structural features that define the cohesin-dockerin interaction. The structure belonging to C. thermocellum revealed that the module X is essential for the stability of the complex. Moreover, for the first time the 3D structure of a cohesin-dockerin complex from B. cellulosolvens was determined. In this complex the dockerin is rotated 180º when compared to other complexes. This gives the cellulosome plasticity. In the final chapters, I present the NMR and X-ray crystallography techniques I used throughout the study. Finally, I draw some general conclusions about all the work done.Fundação para a Ciência e Tecnologia - SFRH/BD/35992/2007, and projects PTDC/QUI/68286/2006, PTDC/QUI-BIQ/100359/2008 and PTDC/BIA-PRO/103980/200
