A Universal, Continuous Assay for SAM‐dependent Methyltransferases

• Published in: Angewandte Chemie International Edition Vol. 62; no. 51; pp. e202313912 - n/a
• Main Authors: Menke, Marian J., Schneider, Pascal, Badenhorst, Christoffel P. S., Kunzendorf, Andreas, Heinz, Florian, Dörr, Mark, Hayes, Martin A., Bornscheuer, Uwe T.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 18.12.2023
• Edition: International ed. in English
• Subjects: Assaying; E coli; Enzyme Catalysis; Escherichia coli - metabolism; Fluorescence; High-Throughput Screening; Homocysteine; Hydrogen Sulfide; Lysates; Methionine; Methylation; Methyltransferase; Methyltransferases - metabolism; S-Adenosylmethionine - chemistry; SAM-Dependent Methyltransferases; Sulphides; Hydrogen Sulfide; High-Throughput Screening; Homocysteine; Enzyme Catalysis; SAM-Dependent Methyltransferases
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202313912

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.