Methanol-driven, one-pot chemoenzymatic S-adenosylmethionine regeneration for ambient cobalamin-dependent methyltransferase reactions

S-Adenosylmethionine (SAM) is a universal methyl donor in diverse biochemical processes, underpinning chemical biology, epigenetics, and biocatalysis. However, the instability of SAM and the O2 sensitivity of many SAM-dependent enzymes, such as cobalamin- and SAM-dependent methyltransferases, limit their practical use under standard laboratory conditions. Here, we devised a one-pot chemoenzymatic cascade that regenerates SAM from methanol under ambient reaction conditions without requiring anaerobic chambers, degassing procedures, or sealed reaction vessels. This system exploits the spontaneous condensation of formaldehyde and tetrahydrofolate while orchestrating the activities of four enzymes: alcohol oxidase, 5,10-methylenetetrahydrofolate reductase, cobalamin-dependent methionine synthase (O2-sensitive), and methionine adenosyltransferase. The cascade efficiently scavenges O2 with alcohol oxidase and 2-mercaptoethanol and drives methyl transfer from methanol to homocysteine via 5-methyltetrahydrofolate. Using the SAM-dependent catechol-O-methyltransferase as a model enzyme, we optimized the efficiency of the bifunctional SAM regeneration system for SAM-dependent methylation reactions. The optimized system supports the synthesis of [19-13C]androgens from estradiol and [13C]methanol by the cobalamin- and SAM-dependent estradiol methyltransferase systems under ambient conditions. Therefore, this nature-inspired chemoenzymatic cascade provides a convenient platform for studying SAM- and cobalamin-dependent methyltransferases and synthesizing 13C-methylated chemicals, DNA/RNA, and peptides, thereby expanding the accessibility of SAM-dependent methylation for synthetic chemistry and biotechnology.