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J Biol Chem. 2016 Sep 23;291(39):20487-502. doi: 10.1074/jbc.M116.716019. Epub 2016 Aug 5.

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.

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From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229.
the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
the Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa, Campo dos Mártires da Pátria, 130, 1169-056 Lisboa, Portugal.
the Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109.
the Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210, and.
the Department of Pediatrics, First Faculty of Medicine, Charles University in Prague and General University Hospital, 116 36 Prague 1, Czech Republic.
From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229,
From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229,


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.


POR deficiency; cytochrome P450; cytochrome P450 oxidoreductase; diflavin oxidoreductase; flavoprotein; membrane protein; protein crystallization; protein stability; reductase

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