Objective The filamentous fungus Trichoderma reesei produces two potent cellobiohydrolases, CBHI and CBHII. The two enzymes have different structures but both have active sites located in enclosed tunnels containing constellations of amino acids that interact with each other and the substrate. Our earlier studies and comparisons of the enzymes with homologous cellulases provide narrow structural constraints for proposed catalytic mechanisms and suggest residues whose mutation will alter pH-dependencies. Our objective is examine and engineer the catalytic mechanisms of T. reesei CBHI and CBHII by state of the art methods in molecular and structural biology and bio-organic chemistry. A detailed model of the interaction of a cellulose chain with these enzymes will be generated by crystallographic analysis of mutant enzymes in complex with natural and modified oligosaccharides. Novel mechanistic probes will be used to establish still unknown details of the mechanisms of the wt enzymes, and to delineate changes of mechanism in the engineered proteins. An exhaustive search of interactions in the active site, carried out by making a range of systematically modified inhibitors and probes, together with complementing mutations in the enzymes will lead to iterative improvement of the active site. This will allow us to raise the pH optima of the two enzymes. Cellulolytic enzymes are already used in e.g. textile and paper industry. Genetic engineering, when combined with structural biology and a solid background in enzymology provides powerful means for learning and improving the enzyme function. In this proposal, all these aspects are included making possible significant advances in the understanding and engineering the catalytic mechanisms of cellulases. Since the same catalytic principles apply to a wide range of other glycosidases which also share similar three-dimensional structures and active site topologies, the results of this work have a wide application potential in enzymatic modification of carbohydrates. Better understanding of these enzyme mechanisms provides new product ideas for the existing biotech industries and may well lead to the development of completely new industries. The results will help European industry to utilise renewable resources in the development of novel biodegradable products and processes, and in future production of renewable energy and raw materials. Fields of science natural sciencesbiological sciencesgeneticsmutationnatural scienceschemical sciencescatalysisnatural scienceschemical sciencesorganic chemistryaminesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymesnatural sciencesbiological sciencesmolecular biologystructural biology Programme(s) FP4-BIOTECH 2 - Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998 Topic(s) 0601 - Structure-function relationships Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator TECHNICAL RESEARCH CENTRE OF FINLAND EU contribution No data Address Tietotie 2 02044 ESPOO Finland See on map Total cost No data Participants (4) Sort alphabetically Sort by EU Contribution Expand all Collapse all Eidgenössische Technische Hochschule - ETH Zürich Switzerland EU contribution No data Address 16,Universitätstrasse 8092 Zürich See on map Total cost No data UNIVERSITY OF MANCHESTER INSTITUTE OF SCIENCE AND TECHNOLOGY United Kingdom EU contribution No data Address SACKVILLE STREET M60 1QD MANCHESTER See on map Total cost No data Universiteit Gent Belgium EU contribution No data Address 35,K.L. Ledeganckstraat 9000 Gent See on map Total cost No data Uppsala University Sweden EU contribution No data Address 3,Husargatan 751 24 Uppsala See on map Total cost No data