A Universal, Continuous Assay for SAM-dependent Methyltransferases

Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates. Identification of novel or improved methyltransferases (MTs) may play a key role in the development of new active pharmaceutical ingredients (APIs), as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms. To identify suitable SAM-dependent MTs, the first universal, fluorescence-based high-throughput MT assay compatible with E. coli cell lysates was developed.image

Automated summary

Researchers have developed a continuous fluorescence-based high-throughput assay for methyltransferase activity, which enables the screening of suitable enzymes for enzyme-catalyzed late-stage functionalization. This assay is compatible with E. coli cell lysates and provides a reliable method to determine methyltransferase activity.