Background: A broad spectrum of human diseases including abnormalities in steroidogenesis is caused by mutations in the NADPH P450 oxidoreductase (POR). POR transfers electrons from NADPH to several small molecules, non-P450 redox partners and all microsomal cytochrome P450 proteins. POR disruption affects all partners with disastrous consequences and POR knock-out mice are embryonically lethal. A number of POR mutations and polymorphisms have been characterized from patients and genome sequencing databases and tested for their abilities to support CYP17A1 and CYP19A1 activities. POR also interacts with drug metabolising CYPs such as CYP3A4 which is responsible for metabolism of about 65% of the drugs in the human liver.
Aim and objective: We aim to evaluate the effect of mutations in POR on steroid and drug metabolising cytochrome P450 activities and study the functional basis of POR deficiency.
Method: We analysed the ability of wild type POR and POR variants (A503V, P284L, P284T and some novel mutants) to reduce ferricyanide, MTT, cytochrome b5, cytochrome c and P450s. POR variants were produced as recombinant N-27 form while P450s and Cytochrome b5 were produced as His-tag recombinant protein and purified by ion-exchange and Ni2+ metal chelate chromatography. Reduction of ferricyanide, MTT and cytochrome c was monitored spectrophotometrically by measuring the change in absorbance. We also tested the interaction of POR variants with P450s using ELISA.
Results and conclusion: Ferricyanide and MTT reduction activity of POR was mildly affected by the mutations. We found varied effect of different POR mutants on CYP17A1, CYP19A1 and cytochrome c reduction activity. However, we observed comparable binding of POR mutants with CYP19A1. Further it would be interesting to study interaction of POR variants with other redox partners. In conclusion, characterization of POR mutants provides valuable genotype-phenotype and structure-function correlation.
Funding: This work was supported by the Swiss National Science Foundation(grant number 134926).
01 - 03 Oct 2015
European Society for Paediatric Endocrinology