Objective The structure/function relationship of cellulases belonging to different families (bacterial and fungal endoglucanases) were to be studied by molecular biology, biochemistry, thermodynamic and crystallographic methods.A number of significant results have been produced during this contract, namely:- determination by X-ray crystallography analyses (partner 1) of the first 3D structure of an endoglucanase of family 5: CelA from Clostridium cellulolyticum.- determination by X-ray crystallographic studies (Partner 6) of several 3D structures of the family 7 endoglucanase from Fusarium oxysporum, in native form and in complexes with: epoxyalkylcellobiosides provided by Novo-Nordisk a/s; cellobiose; and the thiooligosaccharide inhibitors provided by Partner 4.- analysis of the thiocellopentaose complex with EG 1 revealing a significant distorsion of the sugar ring in the -1 subsite as predicted by theory (Partners 4, 6 and Novo).- analysis of the complexes of the family 45 enzymes revealing an extensive loop movement upon substrate binding and a geometry of the cleavage site suited for an optimal binding of a transition state with an elongated glycosidic bond (Partners 4 and 6).- characterization of the spatial arrangement of the different constitutive domains of cellulases by dynamic light scattering, and of the rather extended conformation found for the linker peptide joining the catalytic and the cellulose-binding domains of EGV (partner 4 and Novo).- preparation of a bifunctionalized substrate for the determination of cellulase activity by fluorescence energy transfer (partner 4).- the enzymatic degradation of highly crystalline cellulose characterized by transmission electron microscopy; adsorption of various cellulases on cellulose was followed by immunogold labelling (Partner 4 and Novo).- the synthesis of several potential substrate analogs and of specific inhibitors has been achieved for the determination of molecular aspects of the active site of the cellulases studied within our network (Partners 4, 3);- preliminary observations of the thermal unfolding by differential scanning calorimetry of CelA in different solvent conditions; preliminary studies on hydrolysis of cellulose by CelA (Partner 5);- production of the entire form of CelA (catalytic domain + dockerin); evidence that spontaneous proteolysis of this form can be prevented when the protein is complexed with the cohesin of the scaffolding protein (partner 2); production of large amounts of the catalytic domain of CelC (partner 2); preparation and preliminary studies of a new three-domain cellulase, CelG (partner 2); construction of new mutants of CelA for further structure/function studies in collaboration with Partner 1 (Partner 2);- successful crystallization experiments leading to reproducible crystals, suitable for X-ray analyses, with two cellulases from Clostridium cellulolyticum, CelC (family 8) and CelF (family 48) (Partners 1,2); structural analyses and comparison of active sites of glycohydrolases for a better understanding of the cellulase catalytic machinery (Partners 1, 4, 6). Fields of science natural sciencesbiological sciencesmicrobiologybacteriologynatural sciencesearth and related environmental sciencesgeologymineralogycrystallographynatural sciencesphysical sciencesopticsmicroscopyelectron microscopynatural scienceschemical sciencesanalytical chemistrycalorimetrynatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Programme(s) FP3-BIOTECH 1 - Specific research and technological development programme (EEC) in the field of biotechnology, 1990-1994 Topic(s) Data not available Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator Centre National de la Recherche Scientifique (CNRS) EU contribution No data Address Boulevard Pierre Dramard 13326 Marseille France See on map Total cost No data Participants (4) Sort alphabetically Sort by EU Contribution Expand all Collapse all CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France EU contribution No data Address 601,Rue de la Chimie 601, Domaine Universitaire 38041 GRENOBLE See on map Total cost No data Consiglio Nazionale delle Ricerche (CNR) Italy EU contribution No data Address Corso Europa 30 16132 Genova See on map Total cost No data UNIVERSITY OF YORK United Kingdom EU contribution No data Address University Road YO1 5DD YORK See on map Total cost No data Universiteit Gent Belgium EU contribution No data Address 9000 Gent See on map Total cost No data
