Ein universeller, kontinuierlicher Assay für SAM‐abhängige Methyltransferasen

Abstract

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 l‑homocysteine and H2S to result in a selective fluorescence readout via reduction of a coumarin‐based 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.