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,...

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Published in:Angewandte Chemie International Edition 2023-12, Vol.62 (51), p.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: Article
Language: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
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Summary: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.
ISSN:1433-7851
1521-3773
1521-3773
DOI:10.1002/anie.202313912