Objective While most lipase are active on neutral glycerides only, a few of these enzymes share lipase and phospholipase activity. The objective of this project is to elucidate the structural parameters that determine the interaction of (phospho)lipases with phospholipids and with triglycerides. Such studies should increase our insight in the importance of such interactions for stereopositional- and headgroup specificity of lipases. To this and the construction of hybrid protein, the synthesis of specific inhibitor molecules and the structure determination of inhibited enzymes was foreseen.The construction of active chimeric mutants has been achieved in the laboratories of partners 01, 03 and 04. Participant 04 has produced a chimeric guinea pig lipase related protein (GPLRP2) mutant in which the C-terminal domain was substituted by the corresponding human pancreatic lipase (HPL) domain. The GLRP2 and the GLRP2/HPL hybrid differ in stability, but have similar catalytic properties. The crystal structure of the GPLRP2/HPL hybrid has been solved at 2.1 Å resolution (publication 20).Partner 03 succeeded in the display of active Staphylococcus hyicus (SHL) on the surface of S. carnosus cells; this finding may have implications for future applications of SHL and of lipases in general.In close collaboration participants 01 and 03 continued their efforts to localize the phospholipase domain in SHL. A hybrid of SHL, in which the C-terminal domain has been exchanged with the corresponding domain from S. aureus (SAL) lipase, had lost its phospholipase activity (publication 4). Recent results have localised a limited (60-100 amino acids) domain responsible for phospholipase activity.To facilitate purifications, hexa-histidine tags were introduced into SHL and SAL. Both proteins were made available to participant 05 and have been used in a large screen of about 200 new crystallisation conditions to improve the quality of SHL crystals. Crystals of SHL diffract to a resolution of 2.8 Å and a data set has been obtained. The crystals of SAL are too tiny to collect any diffraction data from.Participant 02 has studied the role of the propeptide of fungal (Rhizopus) lipases on activity and refolding. Moreover, this partner has replaced Leu258, a residue important for stereoselectivity, by Phe and found a 3.5-fold increased ratio of phospholipase to lipase activity. of Rhizopus oryzae lipase (ROL).Participant 01 has synthesised a series of phosphonate inhibitors that have been made available to all other partners. A crystal structure of cutinase inhibited with a tri-hexanoyl analogue has been solved (publication 27) and the structures of Pseudomonas aeroginosa and Chromobacterium viscosum lipase, after inhibition with the tri-hexanoyl-and the tri-octanoyl analogues have been solved by participant 05. Fields of science engineering and technologymaterials engineeringcrystalsnatural sciencesbiological sciencesbiochemistrybiomoleculeslipidsnatural scienceschemical sciencescatalysisnatural scienceschemical sciencesorganic chemistryaminesnatural 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 Rijksuniversiteit Utrecht EU contribution No data Address 8,Padualaan 3584 CH Utrecht Netherlands 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 Chemin Joseph - Aiguier 13402 Marseille See on map Total cost No data Institut für Chemo- und Biosensorik e.V. Germany EU contribution No data Address Mendelstrasse 7 48149 Münster See on map Total cost No data THE UNIVERSITY OF TUEBINGEN Germany EU contribution No data Address Waldhaeuser strasse, 70/8 72076 TUEBINGEN See on map Total cost No data UNIVERSITY OF GRONINGEN Netherlands EU contribution No data Address 4,Nijenborgh 4 6747 AG GRONINGEN See on map Total cost No data