The structural and functional characterisation of 10-15 lipases. The aim of the project is to acquire so much new knowledge about a sufficient number of these enzymes that it will be possible to understand why they are lipases and how they function as such.
The structure of lipases from procaryotic species, in particular Pseudomonas species has been determined and information concerning mechanism of action substrate specificity and inhibitory compounds has been gathered. Crystallographic studies on Pseudomonas glumae lipase has allowed an almost complete native dataset (87% completion) to be obtained at 3 angstroms resolution. Also datasets of 3 heavy atom derivatives one gold, a mercury and a platinum containing compound, werecollected at 3.0 angstroms giving rise to isomorphic changes of 13%, 22% and 12%, respectively. Pseudoglycerides containing ester, ether and amido linked groups were synthesized in optically pure form. These trigylceride analogues have been investigated as substrates for the highly purified lipase from Pseudomonas glumae. The Pseudomonas lipase attacks the 3-ester bond slightly better than the 1-esterbond, but is able to effectively hydrolyze secondary ester bonds as well. Moreover, in the hydrolysis of the secondary ester bond the Pseudomonas glumae lipase appeared to possess a strong stereopreference: the R-isomer is hydrolyzed 50 times more rapidly than S3. For triglyceride substrates the steropreference of a wide range of lipases has been investigated and some lipases have been found to switch preference depending on the acyl chain length.
It is the objective of this project to obtain structural information of lipases from prokaryotic species, in particular from Pseudomonad species, by crystallographic methods. The diversity of primary structures of lipases in general points to a diversity of the 3-dimensional properties of lipases as well. Thus it is important to investigate those lipases that are good representatives of the family of lipases they belong to. Earlier studies have indicated that Pseudomonas lipases constitute such a distinct family, while these enzymes have excellent properties for application in areas ranging from foods to detergents.
The structural information will be used again to gain understanding of lipolytic activity. Functional aspects will be studied using well defined, stereochemically pure substrates and substrate analogs of eg triglyceride molecules containing amide and ether linked fatty acids apart from the normal ester linkages. Kinetic studies in bulk and on those substrates that will form monolayers on water air surfaces will provide detailed information concerning the mode of action of Pseudomonas lipases on organized lipid water interfaces.
Funding SchemeCSC - Cost-sharing contracts
3584 CH Utrecht