Final Activity Report Summary - MANNOSE BINDING PROT (The molecular basis for both selective and flexible carbohydrate protein recognition)
Protein-carbohydrate recognition plays a critical role in many biological processes and the structural basis for the interaction of proteins with complex carbohydrates is poorly understood. The interaction of degradative enzymes with their target substrates within the plant cell wall provides an excellent model system in which carbohydrate protein recognition can be studied. The determination of how proteins may exhibit selectivity for a specific type of polysaccharide, while at the same time displaying relaxed ligand specificity to accommodate the variation in the structure of the target carbohydrate polymers, provides important insights into both the selectivity and flexibility of protein carbohydrate recognition.
An elegant example of this plasticity in ligand recognition is the hydrolytic system that degrades mannose-containing polysaccharides. Mannosidases and their associated carbohydrate binding modules (CBMs) display both remarkable specificity for D-manno-configured substrates but at the same time accommodate, to varying degrees, both homopolymers and heteropolymers. The recognition of mannosides is central to a diverse array of current industrial and medical processes. Thus, in this project two representative family 35 CBMs from Cellvibrio japonicus were studied. Man5C-CBM35 and Abf62-CBM35 binds specifically to mannose and xylose polymers, respectively. The Man5C-CBM35 structure was solved by NMR, which informed a site-directed mutagenesis strategy leading to the identification of the amino acids involved in ligand recognition. The crystal structure of Abf62-CBM35 was also determined, which informed a mutagenesis strategy designed to identify the ligand binding residues. The data show that in family 35 the carbohydrate binding site is situated in a different area than generally found in CBMs. The mannosidase BTMan2A from the human colonic bacterium Bacteriodes thetaiotaomicron was structurally and biochemically characterised and the structure of the transition state was explored. Preliminary data suggest that the geometry of this important structure varies between different mannose degrading enzymes.
Another part of the project focused on engineered hemicellulases. As the major hemicellulose component in plant cell wall is the xylan, and as there is an urgent need to generate thermostable hemicellulases with the appropriate biochemical properties for existing biotechnological processes biochemical and structural studies have been carried out on thermostable xylanases. The data provide insight into the mechanism by which thermostability can be introduced into these industrially important enzymes and, intriguingly, how these biocatalysts can use the side chains of xylans as specificity determinants.
An elegant example of this plasticity in ligand recognition is the hydrolytic system that degrades mannose-containing polysaccharides. Mannosidases and their associated carbohydrate binding modules (CBMs) display both remarkable specificity for D-manno-configured substrates but at the same time accommodate, to varying degrees, both homopolymers and heteropolymers. The recognition of mannosides is central to a diverse array of current industrial and medical processes. Thus, in this project two representative family 35 CBMs from Cellvibrio japonicus were studied. Man5C-CBM35 and Abf62-CBM35 binds specifically to mannose and xylose polymers, respectively. The Man5C-CBM35 structure was solved by NMR, which informed a site-directed mutagenesis strategy leading to the identification of the amino acids involved in ligand recognition. The crystal structure of Abf62-CBM35 was also determined, which informed a mutagenesis strategy designed to identify the ligand binding residues. The data show that in family 35 the carbohydrate binding site is situated in a different area than generally found in CBMs. The mannosidase BTMan2A from the human colonic bacterium Bacteriodes thetaiotaomicron was structurally and biochemically characterised and the structure of the transition state was explored. Preliminary data suggest that the geometry of this important structure varies between different mannose degrading enzymes.
Another part of the project focused on engineered hemicellulases. As the major hemicellulose component in plant cell wall is the xylan, and as there is an urgent need to generate thermostable hemicellulases with the appropriate biochemical properties for existing biotechnological processes biochemical and structural studies have been carried out on thermostable xylanases. The data provide insight into the mechanism by which thermostability can be introduced into these industrially important enzymes and, intriguingly, how these biocatalysts can use the side chains of xylans as specificity determinants.