Autor(en): | Meichsner, Shari L. Kutin, Yury Kasanmascheff, Müge |
Titel: | In-cell characterization of the stable tyrosyl radical in E. coli ribonucleotide reductase using advanced EPR spectroscopy |
Sprache (ISO): | en |
Zusammenfassung: | The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di-iron tyrosyl radical (Y122.) cofactor for activity. The knowledge on the in vitro Y122. structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122.. Here, we characterize this essential radical in whole cells. Multi-frequency EPR and electron-nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y122. are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122. per β2 distribution, supporting the key role of Y. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions. |
Schlagwörter: | EPR spectroscopy Metalloenzymes Ribonucleotide reductase Tyrosyl radicals Unnatural amino acids |
URI: | http://hdl.handle.net/2003/40845 http://dx.doi.org/10.17877/DE290R-22702 |
Erscheinungsdatum: | 2021-04-12 |
Rechte (Link): | https://creativecommons.org/licenses/by/4.0/ |
Enthalten in den Sammlungen: | Physikalische Chemie |
Dateien zu dieser Ressource:
Datei | Beschreibung | Größe | Format | |
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Angew Chem Int Ed - 2021 - Meichsner - In‐Cell Characterization of the Stable Tyrosyl Radical in E coli Ribonucleotide.pdf | DNB | 1.24 MB | Adobe PDF | Öffnen/Anzeigen |
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