Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase

Abstract

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Rational evolution of the cofactor‐binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes

“…However, prior to the present study, none of the mutations located at the FAD domain reported to date has enhanced activity, as documented here for mutants 1‐014 and 1‐053, which supported CYP2C9 and CYP2A6 activity respectively with NADPH more efficiently than the wt (Figs and respectively). A mutation at the FMN domain, Q153R, led to enhanced activity up to 4‐fold with CYP19A1, CYP1A2, CYP2C19, CYP2D6 and CYP3A4 . Similar to our results, the benefit of this mutation was dependent on the P450 form studied and decreased activity was seen with some P450s compared to the wt.…”

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

“…The CYP19A1 deficiency is linked to genetic disorders of estrogen biosynthesis in women and developmental disorders in men [19,31,46,85,86]. Previously we have tested the CYP19A1 variants (R264C and R26H) and POR genetic variants in separate studies that focused on individual genes [59,64,70]. Cytochromes P450 and P450 reductase have a large number of genetic variations and, therefore, many individuals may harbor variations in P450 reductase as well as P450 genes that may alter the enzymatic activities of cytochromes P450 [87].…”

“…The biosynthesis of estrogens requires the aromatase activity of CYP19A1, which is reliant on POR. Figure adapted from our previous publication by [64]. B: Electron transfer from NADPH to CYP19A1 is carried out via POR through reduced FAD and FMN.…”

“…The P284L is a rare variant found mostly in Europeans, and the P284T variant had been only observed in an African population [64,82,83]. The Y607C is a rare genetic variant found in both the Asian and European populations ( Table 2).…”

“…CYP19A1 is a 503 amino acid protein (NP_000094) encoded by the CYP19A1 gene (GeneID:1588, NCBI: NM_000103), located on chromosome 15 (15q21.2, GRCh38 15:51208056-51338597). CYP19A1 is a membrane-bound protein located in the endoplasmic reticulum (ER) and is a member of the may severely affect the production of estrogens, and several variations of POR exist with significant population distribution, which can alter enzymatic activities of CYP19A1 [64][65][66][67][68]. We, therefore, tested the impact of some population variants of POR on estrogen formation by the two variants of CYP19A1.…”