Human
NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal
deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the
protein was less stable than WT. The activities of
cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several
xenobiotic-metabolizing
cytochromes P450, indicating that the A287P
protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT
protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P
proteins were also examined in an osteoblast cell line by treatment with
cycloheximide, a
protein synthesis inhibitor, showing that the level of A287P
protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes
POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent
flavin-binding defect.