High-throughput assays for SAM-dependent methyltransferases: advances, challenges, and future perspectives

Covering: up to January 2026Methyltransferases (EC 2.1.1.-) represent one of the most functionally diverse enzyme families in biology. Found across all domains of life-including bacteria, archaea, eukaryotes, and viruses-these enzymes catalyze a wide range of methylation reactions, commonly on O-, N-, and C-atoms, in a chemo- and regioselective manner. The wide set of substrates methylated is why a universal assay for the methyltransferase's activity screening has not yet been established. Despite this diversity, most methyltransferases utilize a common methyl donor, the S-adenosyl-L-methionine (SAM). This shared feature has enabled the development of several assay strategies based on the detection of S-adenosyl-L-homocysteine, the by-product of SAM-dependent methylation, or its degradation products. Colorimetric and fluorometric assays have been widely employed to monitor methyltransferase activity, each with distinct advantages and limitations, depending on the substrate/product detection of the methylation reaction, but also whether they require purified enzymes or work in crude (E. coli) cell lysates. However, the methylation of substrates with more than one nucleophilic atom requires sophisticated analytical tools for accurate monitoring of the regiospecificity of methyltransferases. This complexity often limits the broader exploitation of methyltransferases, particularly in enzyme engineering efforts, where large datasets are generated and must be efficiently analyzed. This review critically examines the high-throughput screening assays developed to date for methyltransferase activity.