Final Report, FWF Project 22406, Alkylglycerol monooxygenase, cloning and biochemistry
Ernst R. Werner, Divison of Biological Chemistry, Biocenter, Medical University of Innsbruck
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.
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