The transfer of a methyl group is an essential step in many reactions occurring in every living cell. This transformation is catalyzed by methyltransferases (MTases). All MTases share the need for a cofactor to perform their action. S-adenosyl-L-methionine (SAM, AdoMet) is the most frequently used methyl donor in biological systems. Recent studies showed the ability of DNA-C-MTases as well as small molecule-C-MTases to accept SAM analogues for transfer of extended carbon chains to various substrates. Anyway, the improvement towards a broader range of cofactor analogues is of great interest in terms of both biocatalytic synthesis and DNA diagnostics. This proposal aims to combine directed enzyme evolution with rational protein engineering to obtain C-MTases for improved transfer of extended groups from SAM analogues. In general, it is very difficult to develop selection strategies for MTases. The first part of the proposal describes the directed evolution of the DNA-MTase M.SssI towards cofactor analogues by in vitro selection of positive clones. For this purpose the protection of DNA from fragmentation by cognate restriction endonucleases will be used after in vitro expression of gene variants in water-in-oil emulsion droplets (in vitro compartmentalization). This method links genotype and phenotype and provides an effective selection of evolved variants. The result of M.SssI evolution will afterwards be transferred to rational protein engineering of the small molecule MTase NovO. The fact that both enzymes share high structural similarities of their core fold, which include the cofactor binding site, allows the combination of error-prone mutagenesis for directed evolution of M.SssI and site-directed mutagenesis for rational protein design of NovO. Besides analysis of enzyme activities, also biophysical properties of both enzymes will clarify the mechanism of protein-ligand interaction. Therefore dissociation constants of MTases and cofactors will clarify the importance of individual mutations for cofactor specificity. In sum, the proposed work will give new important insights and stimulate prospective applications of alkyl transfer reactions.
|Effective start/end date||1/10/14 → 10/07/15|