Repurposing methyltransferase-cofactor interactions for engineered microbial production of isotopically labelled SAM and SAH

Isotopically labelled S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) are essential cofactors in methylation reactions. They serve as key tools for structural and mechanistic studies of methyltransferases and metabolic flux analysis using NMR spectroscopy and isotope-dilution mass spectrometry (MS). However, uniformly labelled SAM and SAH are not commercially available, and the existing methods of production (enzymatic, chemical or microbial) are costly, technically demanding, or yield mixtures of stereoisomers requiring extensive purification. We have developed a purification strategy that exploits the naturally high-affinity binding of SAM and SAH by selected methyltransferases. His-tagged methyltransferases were overexpressed in Escherichia coli grown on isotope-enriched minimal media, enabling the co-purification of the isotopically labelled cofactors to be co-purified by the standard metall chelate chromatography. Subsequent protein denaturation quantitatively released the bound ligand. This work introduces a simple, robust, and widely applicable platform for the in-house production of isotopically labelled SAM and SAH using standard protein expression and purification equipment. By repurposing high-affinity methyltransferases as disposable affinity carriers, the method eliminates the need for expensive precursors, specialized enzymatic cascades, metabolic engineering, or preparative chromatography, while delivering products of exceptional stereochemical and isotopic purity. The approach is readily adaptable to different isotope-labeling schemes and can be generalized to other tightly bound metabolites, providing a versatile tool for structural biology, enzymology, and metabolic research.