S-Adenosylmethionine-dependent methyltransferases are versatile tools for the specific alkylation of many compounds, such as pharmaceuticals, but their biocatalytic application is severely limited owing to the lack of a cofactor regeneration system. We report a biomimetic, polyphosphate-based, cyclic cascade for methyltransferases. In addition to the substrate to be methylated, only methionine and polyphosphate have to be added in stoichiometric amounts. The system acts catalytically with respect to the cofactor precursor adenosine in methylation and ethylation reactions of selected substrates, as shown by HPLC analysis. Furthermore, H and C NMR measurements were performed to unequivocally identify methionine as the methyl donor and to gain insight into the selectivity of the reactions. This system constitutes a vital stage in the development of economical and environmentally friendly applications of methyltransferases.

S-Adenosylmethionine (SAM)-dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O-methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM-derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l-methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4-O-methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40-70 % yield and high selectivity. The regiocomplementary 3-O-methylation was catalysed by catechol O-methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM-dependent alkylation processes.

Mg -dependent catechol-O-methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol-O-methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S‐Adenosylmethionine (SAM)‐dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi‐enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non‐natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C‐1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

BackgroundMethionine adenosyltransferases catalyse the synthesis of S-adenosylmethionine, a cofactor abundant in all domains of life. In contrast to the enzymes from bacteria and eukarya that show high sequence similarity, methionine adenosyltransferases from archaea diverge on the amino acid sequence level and only few conserved residues are retained.ResultsWe describe the initial characterisation and the crystal structure of the methionine adenosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis. As described for other archaeal methionine adenosyltransferases the enzyme is a dimer in solution and shows high temperature stability. The overall structure is very similar to that of the bacterial and eukaryotic enzymes described, with some additional features that might add to the stability of the enzyme. Compared to bacterial and eukaryotic structures, the active site architecture is largely conserved, with some variation in the substrate/product-binding residues. A flexible loop that was not fully ordered in previous structures without ligands in the active side is clearly visible and forms a helix that leaves an entrance to the active site open.ConclusionsThe similar three-dimensional structures of archaeal and bacterial or eukaryotic methionine adenosyltransferases support that these enzymes share an early common ancestor from which they evolved independently, explaining the low similarity in their amino acid sequences. Furthermore, methionine adenosyltransferase from T. kodakarensis is the first structure without any ligands bound in the active site where the flexible loop covering the entrance to the active site is fully ordered, supporting a mechanism postulated earlier for the methionine adenosyltransferase from E. coli. The structure will serve as a starting point for further mechanistic studies and permit the generation of enzyme variants with different characteristics by rational design.