DEVELOPING NOVEL APPROACHES TO IMPROVE THE NUTRITIVE VALUE OF CEREAL-BASED DIETS FOR POULTRY: THE PLASTICITY/FLEXIBILITY OF TYPE I COHESIN-DOCKERIN COMPLEXES

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Structure and function relationships in novel cellulosomal enzymes and cohesindockerin complexes

Publication . Brás, Joana Luís Armada; Fontes, Carlos Mendes Godinho de Andrade; Prates, José António Mestre

Plant cell walls are the most abundant source of organic carbon on earth, providing an extraordinary supply of energy for various microorganisms. The energetic constrains posed by anaerobic ecosystems lead to the evolution of highly efficient multi-enzymatic complexes, termed cellulosomes, which orchestrate the deconstruction of structural carbohydrates. Clostridium thermocellum cellulosome has been extensively studied as the bacterium exhibits one of the highest growth rates on cellulose. Cellulosomes are assembled by a large non-catalytic multi-modular scaffoldin which contains repeated type I cohesins. Type I dockerin modules, usually located at the C-terminus of enzymes, bind tenaciously to type I cohesins. Scaffoldins may contain a type II dockerin which specifically recognizes type II cohesins located at the cell envelope, allowing the cell surface attachment of cellulosomes. Here a combination of methodologies was applied to study the structure and function relationships of novel cellulosomal enzymes and cohesin-dockerin complexes. Innovative molecular biology and biochemical protocols that can be applied to crystallize and solve the structure of cohesin-dockerin complexes are described in chapter 2. In addition, the crystal structures of two novel type I cohesin-dockerin complexes (CtCohOlpC-Doc124A and CtCohOlpA-Doc918) are described here. They revealed that the two dockerins are unusual since they lack the structural symmetry that supports the dual binding mode typical of type I modules. Thus, these dockerins present a single binding mode and seem to bind preferentially to cohesins located at the bacterium cell surface and not to cellulosomes (chapter 3). Doc124A is the dockerin of CtCel124A, an endoacting cellulase with a superhelical fold that acts in synergy with the major cellulosomal exo-cellulase, Cel48S, during cellulose hydrolysis. The crystal structure of CtCel124A in complex with two cellotriose molecules suggests that the enzyme may target the interface between crystalline and amorphous cellulose (chapter 5). In addition, the structure of a novel type II cohesin-dockerin complex (CtCohScaC2-XDocCipB) was solved. The functional importance of specific dockerin residues was determined. Type II dockerins are suggested to present two different cohesin-binding faces that express different specificities (chapter 4). Finally, the crystal structure of a penta-modular cellulosomal protein (CtXyl5A), previously of unknown function, was assessed (chapter 6). This protein is one of the largest cellulosomal components and comprises a GH5, two CBMs from families 6 and 13, a fibronectin type III-like module, a CBM from family 62 and a type I dockerin. CtGH5 has a canonical (α/β)8-barrel fold and displays specificity for arabinoxylans and as such, is defined as an arabinoxylanase. CtCBM6 adopts a β-sandwich fold and displays affinity for the reaction products generated by CtGH5 and for undecorated xylooligosaccharides. In addition, the penta-modular structure revealed a great flexibility for the CtCBM62 domain.

2012-11Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

PIDDAC

Número da atribuição

SFRH/BD/38667/2007