Glyceryl Ether Monooxygenase

Glyceryl-ether monooxgenase is an enzyme activity found throughout the body of mammals, with highest activities in the liver. It cleaves glyceryl-ether lipids to glycerol and the respective aldehyde by a monooxygenase type reaction with the aid of tetrahydrobiopterin, which is essential for the reaction. Etherlipids are known to be membrane constituents, but also can serve as signal mediators. Tetrahydrobiopterin is a low molecular weight cofactor of aromatic amino acid hydroxylases and nitric oxide synthases. Tetrahydrobiopterin shows structural similarity to the vitamins folic acid and riboflavin, but is biosynthesized in mammals from guanosine 5` triphosphate. Although glyceryl-ether monooxygenase activity has been repeatedly detected by several independent research groups, no sequence has been associated with this enzyme activity so far, and hence no sequence and no position of the gene in the human nor in another mammalian genome has been assigned. In addition, the physiological role of the glyceryl-ether monooxygenase has not been studied yet. The aim of the present project is to assign a sequence in the almost complete mammalian genomes to this enzyme, and to use this information to study the biochemistry, physiological role and potential association to diseases with unknown genetic background. For this purpose we plan to purify the glyceryl-ether monooxygenase protein in its active form, and to determine its sequence. To enable a successful purification of the enzyme, we have developed a novel, rapid and sensitive enzyme assay on the basis of a fluorescent substrate kindly designed and provided by Albin Hermetter, University of Technology, Graz, Austria. In order to unravel the physiological significance of glyceryl-ether monooxygenase, we plan to study mice with a mutation in the gene encoding this enzyme, and to study the effects of downregulation and overexpression of the respective gene in cultivated mammalian cells. We also plan to generate high amounts of pure protein by recombinant expression techniques to study its structure- function relationships, with special emphasis on the role of the tetrahydrobiopterin cofactor in the reaction.

Aim of Project 19764 was to assign a sequence to a the only mammalian enzyme known to cleave a special class of lipids, the glyceryl ethers. We were able to solve this almost 50 year old problem by a combination of bioinformatic database searches and molecular cell biology techniques. As a result of the mammalian genome projects thousands of novel proteins have been predicted, but no function of these had been known. Our results assign a function to one of these predicted proteins and improve our functional understanding of the mammalian genome. In addition, they provide the basis for future studies of biochemistry and function of glyceryl ether monooxygenase using the full spectrum of modern molecular biological tools. Glyceryl esters, which in the form of triacyl-glycerols (the so-called triglycerides) serve as major energy store, are also important as membrane components and signalling molecules. In glyceryl ethers, in contrast, side chains are bound to glycerol by an ether rather than an ester linkage. Glyceryl ether lipids have not been studied as well as glyceryl ester lipids. Nevertheless, ether lipids were found to be as diverse and as widespread as glyceryl ester lipids. Glyceryl ether lipids are already known to serve functions in signal transduction, inflammatory response, sperm development and protection of the eye from cataract. Glyceryl ether monooxygenase may regulate the concentration of these lipids in the body and may therefore serve an important physiological function. Its sequence had not been assigned for a long time since it is a membrane protein which is too labile to be purified. We developed a novel method which allowed determination of glyceryl ether monooxygenase activity in very small amounts of material. This enabled us to monitor the enzyme activity in mammalian cells treated with candidate genes. Bioinformatic tools then selected ten candidates out of the more than 20.000 mammalian genes, which comprised a gene encoding glyceryl ether monooxygenase activity. Glyceryl ether monooxygenase is one of only five enzymatic reactions known to require tetrahydrobiopterin. This compound has structural similarities to the vitamins folic acid and riboflavin. In contrast to these vitamins which have to be taken up with the diet, tetrahydrobiopterin is synthesised by animals. Tetrahydrobiopterin is currently in use to treat a metabolic disorder (phenylketonuria) and in development to treat vascular dysfunction in humans. The results of our research will enable the preparation of tools to learn about treatment effects of tetrahydrobiopterin related to glyceryl ether lipid metabolism. It also defines glyceryl ether monooxygenase as a third, distinct type of tetrahydrobiopterin-dependent enzyme.