Molecular mechanisms and roles of the mammalian 2-oxoglutarate-dependent oxygenases AlkBH1 and AlkBH7

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Independent from the DNA genetic code, gene and protein activity is controlled by epigenetic mechanisms through writing, reading and erasing specific epigenetic marks. These marks include the covalent modifications of histones, DNA and RNA with methyl groups. These methyl groups play an essential role in many cellular processes. Responsible for the spacial and temporal maintenance of these epigenetic marks is carried out by so called "writer" and "eraser" enzymes. Here enzymes belonging to the Fe2+- and 2-oxoglutarate (2OG; = α-ketoglutarate) dependent oxygenase family are particular prominent actors. About 70 members of the Fe(II)/2OG-family across all kingdoms of life are known to date. These enzymes use the cosubstrate 2OG to bind to ferrous iron and to activate molecular oxygen in order to catalyse the transfer of the molecular oxygen onto a methyl group, resulting either in a stable hydroxylated product (=hydroxylases) or demethylation of the substrate via oxidative demethylation (=demethylases). The subfamily this proposal addresses are the AlkB-homologs (AlkBHs), which are found in various organisms such as Escherichia coli, Drosophila melanogaster, Schizosaccharomyces pombe, Cenorhabditis elegans and mammals.In mammals there are nine AlkB-homologs known by now: AlkBH1-8 and the fat mass- and obesity-associated gene product (FTO). The differences in sequence, structural elements and accessory domains account for their remarkably large diversity in substrates and biological roles. For individual mammalian AlkBHs it was shown, that loss-off function as well as their aberrant expression and activities can be linked to various diseases phenotypes, such as obesity, severe sensitivity to inflammation, multiple malformations, infertility and cancer, which emphasizes the importance of these enzymes. However, a direct link between the molecular functions, observed phenotype and the substrate scope for many of the human AlkBHs is still unclear. In the here proposed research project our aim is to elucidate the molecular mechanism of the most elusive human AlkBHs. We want to learn about their substrates and interaction network. Our focus will be the human AlkBH1 and AlkBH7 proteins. The employed methods involve cell biology, mass spectrometry and structural biology.