Dokument: Development of a Continuous, Uncoupled Fluorescence‐based Assay for Monitoring Methyltransferase Activity: Methylation Going Sour
| Titel: | Development of a Continuous, Uncoupled Fluorescence‐based Assay for Monitoring Methyltransferase Activity: Methylation Going Sour | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=73184 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20260505-125647-2 | |||||||
| Kollektion: | Publikationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Texte » Artikel, Aufsatz | |||||||
| Medientyp: | Text | |||||||
| Autoren: | Chapple, Benjamin Panagiotis [Autor] Böhmer, Lisa M. [Autor] Pietruszka, Jörg [Autor] | |||||||
| Dateien: |
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| Stichwörter: | natural products , enzyme assay , fluorescent probes , high-throughput screening , methyltransferase | |||||||
| Beschreibung: | Methyltransferases (MTs) are versatile biocatalysts capable of regio-, stereo-, and enantioselective methylation of molecules, making them particularly valuable for late-stage functionalization of natural products and their analogues. To further broaden the applicability of these enzymes, it is essential to gain knowledge about their mode of catalysis and develop effective enzyme engineering and screening strategies. This, in turn, requires the development and application of robust analytical techniques, especially when dealing with large-scale screenings, such as those involved in mutagenesis campaigns. In this study, we have established the first uncoupled, continuous, fluorescence-based, high-throughput assay that serves as a close-to-universal system for activity determination of S-adenosyl-l-methionine-dependent MTs. The assay is based on proton release during the methylation, and its use is not limited to purified enzymes, but can also be applied with cell-free extracts and, in certain cases, pretreated whole cells. To validate its applicability, activities of three distinct MTs with different substrate acceptor atoms (C, N, and O) were determined, and the results were benchmarked against HPLC analysis. Additionally, the assay’s utility was demonstrated through a substrate scope screening and the determination of kinetic parameters. Finally, the Z’-factor was evaluated for two different potential enzyme setups to assess the assay’s suitability for high-throughput applications. | |||||||
| Rechtliche Vermerke: | Originalveröffentlichung:
Chapple, B. P., Böhmer, L. M., & Pietruszka, J. (2026). Development of a Continuous, Uncoupled Fluorescence‐based Assay for Monitoring Methyltransferase Activity: Methylation Going Sour. ChemCatChem, 18(3), Article e01714. https://doi.org/10.1002/cctc.202501714 | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 05.05.2026 | |||||||
| Dateien geändert am: | 05.05.2026 |
