Alkylglycerol monooxygenase: Cloning and biochemistry

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.

The aim of the present project was to assign a coding sequence to alkylglycerol monooxygenase, an important enzyme in the metabolism of a class of lipids in our body, the ether lipids. The enzymatic reaction it carries out had been known since 1964. It had not been known, however, which coding sequence in our genome is responsible for this enzyme. Enzymes for which no sequence is known are called orphan enzymes. For humans, about a hundred enzymes with unknown coding sequence, i.e. orphan enzymes, have been described. For many enzymes the sequence had been obtained by isolating it in pure form, and then determining the sequence of amino acids of the pure protein. This was not possible in this case, since alkylglycerol monooxygenase is an especially labile protein embedded in cellular membranes which loses its enzymatic activity quickly when it is taken out of its membrane environment in attempts to isolate it. To solve this problem, we took advantage of the detailed knowledge of virtually all protein-coding sequences which resulted from the human genome characterizing efforts. We anticipated that one of the thousands of protein coding genes in our genome for which the function is not yet known must be responsible for alkylglycerol monooxygenase. Since the methods we had generated to test experimentally whether a gene is responsible for this reaction allowed only the analysis of a few genes, we had to select candidate genes from the human genome and proteome databases. We did this by looking for protein motifs related to the function of the protein. We looked for a certain combination of amino acids known to occur in enzymes which cleave lipids in a way reminiscent of alkylglycerol monooxygenase, the so-called fatty acid hydroxylase motif. We then found in the human proteome database a few proteins with unknown function which contained this motif. When we tested these experimentally, we found that one of them, previously called transmembrane protein 195, was indeed responsible for alkylglycerol monooxygenase activity. This fundamental breakthrough made alkylglycerol monooxygenase accessible to state of the art research. It opened up the way to use the tools of modern molecular biology to study the biochemistry of the enzyme, to manipulate its activity in model organisms in order to study its physiological role, and to browse data bases for disease associations in humans.