Efficient Enzymatic Synthesis and Cofactor Regeneration of Biomimetic S -Adenosyl- l -methionine for Enzymatic Methylation

S -Adenosyl- l -methionine (SAM) is an essential biological cofactor. However, its application in vitro is hindered by high cost and instability. This study addresses this limitation by developing a biomimetic SAM analogue, S -adenosyl- l -methylcysteine (SAMC), and an efficient enzymatic regeneration system. We established an ATP-independent synthesis of its precursor, S -adenosyl- l -cysteine (SAC), using S -adenosyl- l -homocysteine (SAH) hydrolase. Structural analysis and subsequent directed evolution of thiopurine methyltransferase from Aspergillus clavatus ( Acl TMT) and halide methyltransferase from Arabidopsis thaliana ( At HMT) yielded positive mutants, notably Acl TMT_R43A-T165M and At HMT_Q49A-V140M, which exhibited dramatically enhanced activity (33.1- and 27.1-fold increases in specific activity, respectively) and catalytic efficiency (35.5- and 12.8-fold higher k cat / K M, respectively) for SAC methylation using artificial donors MeOTs and CH 3 I, respectively. Crucially, we constructed a one-pot cofactor regeneration cycle coupling these engineered methyltransferases (MTs) with natural product MTs (MT-6 from Streptomyces cinnamoneus and MT-31 from Nostoc sp.). This system successfully synthesized various methylated flavonoids, quinone, and indole with high conversion rates (exceeding 80% in multiple cases) and identical regioselectivity compared to SAM-dependent reactions. Our work demonstrates that SAMC can functionally replace SAM, providing a cost-effective and efficient platform for in vitro methylation biocatalysis and expanding the toolbox for natural product diversification.