Please use this identifier to cite or link to this item: doi:10.22028/D291-29466
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Title: Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s
Author(s): Bakkes, Patrick J.
Riehm, Jan L.
Sagadin, Tanja
Rühlmann, Ansgar
Schubert, Peter
Biemann, Stefan
Girhard, Marco
Hutter, Michael Christopher
Bernhardt, Rita
Urlacher, Vlada B.
Language: English
Title: Scientific reports
Volume: 7
Issue: 1
Publisher/Platform: Nature Publishing Group
Year of Publication: 2017
Publikation type: Journal Article
Abstract: Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS)n or rigid ([E/L]PPPP)n linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP)5. Molecular dynamics simulations demonstrated that ([E/L]PPPP)n linkers are intrinsically rigid, whereas (GGGGS)n linkers are highly flexible and biochemical experiments suggest a higher degree of separation between the fusion partners in case of long rigid P-linkers. The catalytic properties of the individual redox partners were best preserved in the YR-P5 construct. In comparison to the separate redox partners, YR-P5 exhibited attenuated rates of NADPH oxidation and heme iron (III) reduction, while coupling efficiency was improved (28% vs. 49% coupling with B. subtilis CYP109B1, and 44% vs. 50% with Thermobifida fusca CYP154E1). In addition, YR-P5 supported monooxygenase activity of the CYP106A2 from Bacillus megaterium and bovine CYP21A2. The versatile YR-P5 may serve as a non-physiological electron transfer system for exploitation of the catalytic potential of other P450s.
DOI of the first publication: 10.1038/s41598-017-10075-w
URL of the first publication:
Link to this record: hdl:20.500.11880/28308
ISSN: 2045-2322
Date of registration: 15-Nov-2019
Faculty: NT - Naturwissenschaftlich- Technische Fakultät
Department: NT - Biowissenschaften
Professorship: NT - Prof. Dr. Volkhard Helms
Collections:UniBib – Die Universitätsbibliographie

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