Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug-metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia that may resemble bone malformations resembling Antley-Bixler syndrome. Genome sequencing studies have revealed the existence of a POR missense variant P228. We aimed to determine the detailed functional impact of POR variant P228L for its role in human metabolism. We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates and cytochrome P450 proteins. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed. The single point mutation P228L had reduced thermal stability indicated by a lower melting point compared to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR compared to WT. Enzymes in liver matabolize many different steroids and drugs and their reduced function may affect several metabolic pathways. Metabolism of steroids by CYP17A1 was also affected, indicating disease-causing potential. A single change in the amino acid sequence can affect protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.
15 Sep 2022 - 17 Sep 2022