A Universal,Continuous Assay for SAM-dependent Methyltransferases |
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| Authors: | M Sc Marian J Menke Dr Pascal Schneider Dr Christoffel P S Badenhorst Dr Andreas Kunzendorf B Sc Florian Heinz Dr Mark Dörr Dr Martin A Hayes Prof Uwe T Bornscheuer |
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| Institution: | 1. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany;2. Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, 43183 Mölndal, Sweden |
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| 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 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. |
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| Keywords: | Enzyme Catalysis High-Throughput Screening Homocysteine Hydrogen Sulfide SAM-Dependent Methyltransferases |
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