SUN-424 Impact Of Three Variants In The FAD-Binding Domain On Cytochrome P450 Oxidoreductase Activity

Abstract Disclosure: I.S. Barata: None. E. Lucato: None. M. Rojas Velazquez: None. A.V. Pandey: None. NADPH:cytochrome P450 oxidoreductase (POR) is the electron provider to microsomal cytochrome P450 (CYP) enzymes, involved in a wide number of metabolic pathways, from steroidogenesis to retinoic acid and vitamin metabolism. POR is also crucial for detoxification pathways and may contribute to carcinogen bioactivation either by direct small molecule reduction or through CYP-mediated activity. Therefore, human variants in POR can manifest phenotypically with a very broad phenotype, from impaired steroidogenesis to congenital bone malformations. POR is a single-pass membrane protein composed of a NADPH-binding domain and FAD and FMN-binding domains, linked by a hinge region. Variations in different regions of the protein contribute to the heterogeneous effects of POR variants, and mutations in amino acid residues involved in cofactor binding are commonly associated with deleterious effects. We report three POR variants, Val334Ile, Ser460Tyr, and Val472Leu, located in the FAD-binding domain. The variants were expressed in bacterial systems, partially purified and assayed for their ability to act as electron donor to different CYP enzymes or directly to small molecules. Val334Ile and Val472Leu had decreased CYP2C9, CYP2C19, and CYP3A4 activities, more pronounced for the Val334Ile variant, which maintained only approximately 20% of CYP2C9 and CYP2C19 activity and 70% of CYP3A4 activity. Moreover, preliminary data suggests a pronounced effect for the Val334Ile POR variant in the steroidogenic activity of CYPs 17A1 and 21A2, with approximately 35% of activity compared to wild-type POR. Cytochrome c and resazurin reduction by POR were also impaired for the three variants, ranging from 20 to 65% of wild-type activity. In silico predictions of stability and conservation from ProtVar, DynaMut, MAPP, Poly-Phen-2, PANTHER and other prediction tools were in accordance with the experimental data, predicting a more moderate effect for the Val472Leu variant. Presentation: Sunday, July 13, 2025