English Abstract, FWF Project 22406, Alkylglycerol Monoxygenase: Cloning and Biochemistry,
Ernst R. Werner, Division of Biological Chemistry, Biocenter, Medical University of Innsbruck
A wide variety of lipids with a glycerol backbone occur in the human body. While those glycerol derivatives with attachment of carbon chains via an ester bond are well studied, little is known about lipids containing an ether linkage. They are abundant in the mammalian body, and serve important roles such as nerve development, sperm maturation or prevention of cataract formation.
Alkyl glycerol monooxygenase (glyceryl ether monooxygenase, E.C. 1.14.16.5), is the only known enzyme that cleaves the ether bond in ether lipids, thus serving a crucial step in the degradation of these compounds. So far, no sequence of this enzyme is known. This fact is a major obstacle in the research on this enzyme.
The aim of the present project is to assign a sequence to alkylglycerol monooxygenase, and thereby open the way for state of the art research on the biochemistry and physiological role of this enzyme. In the preceding project 19764 we have successfully completed all essential steps required for performing the sequence assignment. We have developed a highly sensitive, novel assay for precise quantification of alkylglycerol monooxygenase activity in minute amounts of tissue and developed methods for reliable extraction of the activity from cultivated cells. For the assignment of the sequence, we plan to use function expression screening in Xenopus laevis oocytes. This method has been successfully applied to many membrane proteins and membrane enzymes that had not been possible to purify. We have already managed to complete the first essential step of this technique. We were able to reproducibly generate a tetrahydrobiopterin dependent alkylglycerol monooxygenase activity in Xenopus oocytes by injection of rat liver messenger ribonucleic acid. Using standard techniques, the messenger ribonucleic acid will now be fractionated and ultimately yield the sequence encoding alkylglycerol monooxygenase.
After successful completion of the sequence assignment, we want to study the biochemistry of the enzyme in detail. We plan to recombinantly express the protein, and determine which amino acid residues in the sequence are essential for the enzymatic activity and for binding of the cofactor tetrahydrobiopterin. In addition, we plan to characterise binding partners of the enzyme by using two techniques, the split ubiquitin yeast system, and co-purification of proteins with tagged alkylglycerol monooxygenase by affinity chromatography.
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