| Popis: |
Two of the central building blocks of life are proteins and carbohydrates. The interactions between these two disparate biomolecules playa key role in numerous biological processes. Carbohydrates are a major component of the plant cell wall, which consists of a complex network of polysaccharides. The complex structure of the plant cell wall is highly inaccessible to enzyme degradation. To overcome their limited accessibilirf to polysaccharides, microbial cellulases and hemicellulases have developed complex molecular architectures generally comprising both catalytic modules and non-catalytic carbohydrate b1...''1ding modules (CBMs). CBMs display considerable variation L.''1 primary structtlte and are grouped into 51 sequence-based families collated in the continuously updated carbohydrate-active enzyme data base CAZy. Family 35 CBMs provide an interesting example of how the fme-tuning of these modules can have a profound influence on polysaccharide recognition. The ligand specificity of fom members of this CBM family was determined. A CBM35 derived from a modular pectate lyase, Pell0A-CBM35, binds specifically to the unsaturated ends of poly galacturonic acid (.6.GaLL\.-GalA), while QAbf62.l\.CBM35 and CtCBM3Sb display affinity for glucuronic acid and .6.GalA-GalA. A fomth CBM35, CtCBM3Sa, is the only one of these proteins to recognise galactose. The crystal structures of these four CBM35 have been solved, while the structure of QAbf62A-CBM35 was also determined in complex with glucmonic acid. Structure comparison of the four CBM35s, and directed-mutagenesis studies of Pe110A-CBM35 and QAbf62A-CBM35, revealed that the binding sites of the four protein modules exhibit slight differences in the positioning of the key residues, allowing the CBMs to accommodate different ligands. Finally, as demonstrated in a previous study on QAbf62A-CBM35, ligand recognition by Pell0A-CBM35 and CtCBM35a is calcium dependent. This thesis also presents the characterisation of two novel families of CBM L.''lat display new ligand specificities. A member of a Clostridium thermol'ellum family 52 CBM, CtCBM52, was shown to bind selectively to galactopyranose and arabinofuranose, with preference for the galactopyranose sidechain of xyloglucan. This was supported by the crystal structure of CtCBM52 in complex \.viL.''l xyloglucan oligosaccharide, 61-tX-D-galactosyl mannotriose and arabinobiose. The putative role of CtCBM52 in the formation of a polysaccharide lattice that engulfs the host bacterium is discussed. The X14 modules present in several xylanases, including QXynl1A, represent a family of non-catalytic modules whose function was previously unknown. It is shown in this thesis that a member of this family, X14-BD7340, binds to a range of different polysaccharides that include galactans, glucomannans, ~-glucans and AJlans, \.v1.th similar affinities. The crystal structure of X14-BD7340 and site-directed mutagenesis provided insight how this module displays such plasticirf in ligand recogrutlon. The complete degradation of the plant cell wall requires enzymes that are able to release decorations from the backbones of many polysaccharides, such as acetyl groups. This thesis reveals that four family 2 carbohydrate esterases, CtCE2, CjCE2A, CjCE2B and CjCE2C, displayed activity against 4-nitrophenyl acetate, glucomannan, galactoglucomannan and acetylated xylan. Intriguingly, CtCE2, the only CE2 member to be linked to a second catalytic module, which displays cellulase activity, is inhibited by cellulosic polymers. The crystal structure of CtCE2, in complex with cellohexaose, and the apo forms of CjCE2A and QCE2B revealed the structural basis for the observed non-competitive inhibition of CtCE2 by cellulose. The enzyme contains three planar aromatic residues lirJng the deep active site cleft that stack against the glucopyranose rings of cellulose. The biological significance for the dual CBM-esterase activity 1...''1 relation to the appended cellulase, and in the wider context of plant cell wall degradation, is discussed. The crystal structures of two of the CE2 enzymes showed that these serine esterases did not display the canonical catalytic triad; the position of the histidine that activates the serine nucleophile of these enzymes is stabilized through hydrogen bonds with polar backbone carbonyl and amine moieties. |
