Eine biochemische Domino-Reaktion zur Modifikation von biologischen Nukleophilen mit Methyltransferasen

  • A biochemical Domino Reaction for modification of biological nucleophiles with methyltransferases

Dankers, Andreas Johannes; Weinhold, Elmar (Thesis advisor); Albrecht, Markus (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019


The main methyl donor in biological systems is S-Adenosyl-L-methionin (AdoMet). It is naturally synthesized from adenosine triphosphate (ATP) and L-methionine by methionine adenosyltransferases (MAT) and is used by AdoMet dependent methyltransferases (MTases) for sequence-specific methylation of biological nucleophiles. Since the natural methyl group is a poor reporter group, AdoMet analogues with extended and functionalized are synthesized to enable a better detection and additional chemical labeling. In the first part of this thesis, the cofactor binding pocket of the CpG specific DNA-MTase M.MpeI is enlarged by site directed mutagenesis method. This enzyme variant is able to bind synthetic AdoMet analogues with an extended and functionalized site chain for alkylation of cytosine in C5 position in the CpG dinucleotide. That’s why the enzyme variant is a very powerful tool for epigenetic science. In comparison with commercially available CpG-specific DNA MTase M.SssI, the M.MpeI offers advantages especially in loss-free storage over a long period of time and higher expression yields. The reactivity of both enzyme variants with AdoMet analogues is very similar. Synthetic AdoMet analogues are often very instable and disintegrate very fast in the reaction mixture. In addition, the chemical synthesis also provides the biologically inactive epimer. Therefore, in the second part of this thesis, a biochemical domino reaction is designed for compensation of these disadvantages. In the first step, only the biologically active AdoMet analogue is synthesized in situ from ATP and a functionalized L-methionine analogue with a propargyl instead of a methyl group by the human MAT before it`s immediately consumed by a DNA or protein MTase. The L-methionine analogues used in this reaction were successfully synthesized from L-methionine or the selenium derivative L-selenomethionine in a chemical reaction. In this thesis, we succeeded for the first time in modifying DNA and protein substrates with a propargyl group in a biochemical domino reaction using various DNA (including the M.MpeI) and protein MTases. The successful modification of the biological substrates could be validated with different methods.