Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

Abstract

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

Fig. 1.

Structure of human CYPOR and interactions between FAD and polypeptide. (A) Overall structure. The molecule consists of three domains, the FMN domain (blue), the connecting domain (gray), and the FAD/NADP(H) domain (green). Cofactors (FMN, blue; FAD, brown; and NADP⁺, red) are shown as stick models and mutation sites are marked with pink balls. Residue H621 (pink arrow) in rat CYPOR is missing in the human enzyme. (B) Interactions between FAD and the polypeptide observed in human CYPOR structure. Hydrogen bonds and salt bridges are shown with dotted lines and eye lashes indicate hydrophobic interactions. The FAD ring is sandwiched between W679 at the re-face and Y459 at the si-face. The dimethyl benzene ring of the isoalloxazine ring is in close contact with the FMN, and the pyrimidine side of the ring makes a tight hydrogen-bonding network with the polypeptide. The pyrophosphate group of FAD makes salt bridges with R457 and hydrogen bonds with the main-chain amide nitrogen of the peptide segment including V492–T494.

Fig. 2.

SDS-PAGE analysis of the limited trypsin digestions of V492E, R457H, and wild type of CYPOR. The protein: trypsin ratio by weight was 100∶1, except for lanes with asterisks performed at a ratio of 1,000∶1. At indicated times, aliquots were taken for SDS-PAGE analysis. Molecular weight markers in V492E digestion are 1, 100 kDa; 2, 75 kDa; 3, 50 kDa; 4, 37 kDa; 5, 25 kDa; 6, 20 kDa; and in R457H or wild-type CYPOR digestion are 1, 70 kDa; 2, 50 kDa; 3, 40 kDa; 4, 30 kDa; 5, 25 kDa. N-terminal sequence analyses of V492E trypsin digestion products yielded GSGMV67… for band a; and 488INKGEAT… for band b, indicating that the first major cleavage site in V492E was at R487.

Fig. 3.

The β-flap in molecule B of V492E is disordered. Electron densities in the vicinity of FAD and E492 in molecule A (A) and molecule B (B) of the V492E structure. The omit (F_o - F_c) map electron density was contoured at 2.5σ level. The conformations of E492 are different in the two molecules. The densities for the ribityl-adenine portion of FAD and the β-loop-β region (V479–G491; shown in green ribbon) are well defined in molecule A, whereas those in molecule B are barely traceable.

Fig. 4.

UV-vis circular dichroism spectroscopy of CYPOR variants. The CD spectrum of R457H ([protein] = 60 μM in MOPS buffer) was collected in the near UV-vis range. For comparison, the normalized spectra of WT and V492E (14), and of Y181D (16), are shown. Y181D is included to demonstrate the general spectral characteristics of CYPOR-bound FAD, because Y181D contains negligible FMN levels. The sum of Y181D + V492E spectra is also shown for comparison to WT. The contribution of bound FMN (represented by the spectrum of V492E) was subtracted from WT and R457H spectra (see Inset) in order to demonstrate the diminished FAD content of R457H, as indicated by decreased CD at approximately 375 nm.