Speaker
Description
NADPH cytochrome P450 oxidoreductase (POR) is an essential redox protein that functions as an electron donor and central regulator of over 50 partner proteins, including the cytochrome P450 enzyme network (1). While naturally occurring mutations in POR are known to disrupt steroidogenesis and drug metabolism, the molecular mechanisms by which POR sequence variations influence structural dynamics, protein stability, and partner selectivity remain poorly understood (2, 3). To address this gap, we are utilizing computational models to engineer POR variants with optimized stability and specificities towards individual redox partners.
In the initial phase, we designed five novel POR variants for improved overall protein stability using human POR sequence as a template. These sequence variants were expressed in a recombinant Escherichia coli system and purified for functional analysis. Preliminary characterization using Cytochrome C reduction assays confirmed that the engineered proteins retain their fundamental function.
Ongoing and future investigations are focused on assessing biophysical properties like protein stability using a combination of urea denaturation, FastPP thermal stability, and dye-binding thermofluor assays. To understand how these stabilizing mutations impact functional plasticity, the variants will be screened for their efficiency in supporting the catalytic activities of essential steroid and drug-metabolizing cytochrome P450 enzymes. By correlating computational predictions with in vitro thermodynamic and kinetic data, this work aims to establish a framework for creating robust, customized electron transfer partners for both pharmacological research and complex biotechnological applications.
Acknowledgements: Funded by the Swiss National Science Foundation (SNF) grant 204518 and SNF-COST Projects in Switzerland 229294 to Amit V Pandey.
References:
1. Pandey et al.: Pharmacol Ther. 2013;138(2):229-254.
2. Flück et al.: Nat Genet. 2004;36(3):228-230.
3. Jensen et al.: Nat Commun. 2021;12(1):2260.