Biocatalytic cobalt methyl transfer for de/methylation

Despite being rather small in mass and size, respectively, introduction or removal of a methyl moiety in an organic molecule can have considerable impacts on its physical and pharmacological properties. Therefore, methylation and demethylation are highly targeted reactions in terms of the industrial syn- thesis of active ingredients, chemical products as well as intermediates. However, conventional chem- ical methods usually rely on the use of hazardous reagents, and the development of more sustainable strategies is strongly desired. Thus, the utilization of bacterial vitamin B12-dependent methyltransfer- ase systems may offer a green alternative. Up to now, only a limited number of respective systems has been characterized so far for demethylation, and their potential for biotechnological purposes still needs to be explored. While the natural methyl transfer reaction involves four different proteins, in this study a novel concept utilizing only two enzymes is investigated. Both demethylation of a donor mol- ecule and methylation of an acceptor are catalyzed by methyltransferase I, while the corrinoid protein serves as shuttle of the methyl group. Since the cobalamin-cofactor is regenerated in the oxygen- sensitive catalytic cycle, it is only necessary in catalytic amounts. The aim of this project is the development and characterization of the reversible methyl transfer cata- lyzed by simplified cobalamin-dependent methyltransferase systems originating from Desulfitobacte- rium hafniense (dhaf), Acetobacterium dehalogenans (vdm) as well as Hyphomicrobium chloro- methanicum (cmu). Besides expression of the respective enzymes, the project focuses on investigation of the substrate scope regarding methylation and demethylation at different chemical functionalities. In order to substantiate the applicability of cobalamin-dependent methyl transfer for the synthesis of pharmacologically active compounds, the biocatalytic formation of the antiepileptic lacosamide and the antidepressant O-desmethylvenlafaxine is pursued as well as mono-methylation of caffeic acid. Furthermore, optimization of reaction parameters, determination of enzymatic selectivity and applica- bility of the enzymatic systems for intramolecular methyl transfer is of high interest. Summarizing, cobalamin-dependent methyltransferase systems can be expected to be highly promis- ing candidates for various biotechnological and synthetic applications achieving methylation and de- methylation reactions in an environmentally acceptable fashion.

Using biocatalysts, thus proteins, for organic chemistry is getting increasing attention. Their use offers significant advantages over conventional chemistry: first of all, they are made from amino acids, thus from renewables and are therefore also biodegradable. Furthermore, the biocatalysts work at mild condition (moderate temperature, atmospheric pressure as well as neutral pH) and in aqueous media. In this project a new method for the transformation of molecules found in lignin but also used as anti-oxidants was developed. For example, the transformation of antioxidants is rather tricky, since these are sensitive molecules (Especially to molecular oxygen but also to extreme pHs). As a possible application, the developed method allowed to prepare the bioactive and important ingredient of olive oil (3-hydroxytyrosol) from inexpensive precursors. Therefore, the development of this biocatalytic methods represents a valuable alternative to perform such important reactions, in sustainable, simple resource efficient and inexpensive manner.