Engineering carbohydrate-active enzymes: specificity and activity remodeled

• Autores: Trevor Addington
• Directores de la Tesis: Antoni Planas (dir. tes.)
• Lectura: En la Universitat Ramon Llull ( España ) en 2009
• Idioma: inglés
• Tribunal Calificador de la Tesis: Ignacio Fita Rodríguez (presid.), Magda Faijes Simona (secret.), Francesc Canals Suris (voc.), Olga Durany Türk (voc.), Víctor Manuel González Miguel (voc.)
• Materias:
  • Química
    • Bioquímica
      • Enzimología
      • Glúcidos
      • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • To understand and modify the secondary cell walls of plants the project group Enzyme Discovery in Hybrid Aspen for Fiber Engineering (EDEN) was founded composed of nine laboratories with funding from the European Commission. The main target of EDEN´s research is to genetically engineer fiber structure in order to produce transgenic trees with modified properties for the pulp and paper industries. In this target framework, the Populus tremula x tremuloides xyloglucan endotransglycosylase (PttXET16A) was selected for in-depth study of its transglycosylase activity catalyzing cleavage and reconnection of xyloglucan molecules, which is proposed to be involved in secondary cell wall morphogenesis. The creation of a family 16 carbohydrate active enzyme -glucanase/XET hybrids were attempted in order to design a chimeric enzyme with one or more of the following altered properties: specificity, activity, and or stability. The two enzymes, Bacillus licheniformis 1,3-1,4--glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase, are members of the same enzymatic family and have highly homologous 3-dimensional structures. However, the enzymes exhibit different activities, one a hydrolase the other a transferase; different specificities, one accepts only linear glcosydic substrates while the other branched substrates; and different stabilities. Hybrid enzyme construction represented an investigational challenge in order to understand what physical characteristics of both enzymes attribute to the specific pattern of activity and specificity observed.Removal of the 1,3-1,4--glucanase major loop resulted in a folded protein which still maintained some β-glucan hydrolase activity. However, no xyloglucan endotransglycosylase-like activity or specificity was observed. Next, point mutations of the β-sheets forming the enzymatic binding site cleft were mutated to resemble PttXET16A residues. The final chimeric protein neither exhibited XET nor β-glucanase activities. Structural analysis by X-ray crystallography revealed a major unexpected structural rearrangement providing a clear insight for further enzyme engineering.