Fig. 5. (A) Optimised cofactor regeneration system for SAM and its analogues. General scheme of the two parallel cyclic systems for l-methionine (yellow shading) and ATP (green shading) resulting in a bicyclic SAM regeneration system. Stoichiometric amounts of MT substrate, SMM, and polyP, and catalytic amounts of cofactor building block (9×) are needed. (B and C) Exemplary HPLC chromatograms for cyclic SAM regeneration assays with RnCOMT and RgANMT with the additional l-methionine regeneration with a maximum of 50 possible turnovers (orange: enzyme assay; grey: control without enzymes). MAT, l-methionine adenosyltransferase; MT, methyltransferase; ADK, adenosine kinase; PPK2-I/II, polyphosphate kinases; SAHH, SAH hydrolase; HSMT, l-homocysteine S-methyltransferase; TTN, total turnover number.