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.

From the Department of Biochemistry University of Texas Health Science Center San Antonio Texas 78229

the Centre for Toxicogenomics and Human Health Genetics Oncology and Human Toxicology NOVA Medical School FCM Universidade Nova de Lisboa Campo dos Mártires da Pátria 130 1169 056 Lisboa Portugal

the Department of Biochemistry Medical College of Wisconsin Milwaukee Wisconsin 53226

the Department of Pediatrics 1st Faculty of Medicine Charles University Prague and General University Hospital 116 36 Prague 1 Czech Republic

the Department of Pharmacology State University of New York Upstate Medical University Syracuse New York 13210 and

the Division of Metabolism Endocrinology and Diabetes Department of Internal Medicine University of Michigan Ann Arbor Michigan 48109

Zobrazit více v PubMed

Pandey A. V., and Flück C. E. (2013) NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol. Ther. 138, 229–254 PubMed

Riddick D. S., Ding X., Wolf C. R., Porter T. D., Pandey A. V., Zhang Q.-Y., Gu J., Finn R. D., Ronseaux S., McLaughlin L. A., Henderson C. J., Zou L., and Flück C. E. (2013) NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology. Drug Metab. Dispos. 41, 12–23 PubMed PMC

Schacter B. A., Nelson E. B., Marver H. S., and Masters B. S. (1972) Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system. J. Biol. Chem. 247, 3601–3607 PubMed

Ono T., and Bloch K. (1975) Solubilization and partial characterization of rat liver squalene epoxidase. J. Biol. Chem. 250, 1571–1579 PubMed

Ilan Z., Ilan R., and Cinti D. L. (1981) Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system. J. Biol. Chem. 256, 10066–10072 PubMed

Shen A. L., O'Leary K. A., and Kasper C. B. (2002) Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome P450 oxidoreductase. J. Biol. Chem. 277, 6536–6541 PubMed

Henderson C. J., Otto D. M., Carrie D., Magnuson M. A., McLaren A. W., Rosewell I., and Wolf C. R. (2003) Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase. J. Biol. Chem. 278, 13480–13486 PubMed

Gu J., Weng Y., Zhang Q.-Y., Cui H., Behr M., Wu L., Yang W., Zhang L., and Ding X. (2003) Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase. J. Biol. Chem. 278, 25895–25901 PubMed

Flück C. E., Tajima T., Pandey A. V., Arlt W., Okuhara K., Verge C. F., Jabs E. W., Mendonça B. B., Fujieda K., and Miller W. L. (2004) Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome. Nat. Genet. 36, 228–230 PubMed

Arlt W., Walker E. A., Draper N., Ivison H. E., Ride J. P., Hammer F., Chalder S. M., Borucka-Mankiewicz M., Hauffa B. P., Malunowicz E. M., Stewart P. M., and Shackleton C. H. (2004) Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study. Lancet 363, 2128–2135 PubMed

Panda S. P., Guntur A. R., Polusani S. R., Fajardo R. J., Gakunga P. T., Roman L. J., and Masters B. S. (2013) Conditional deletion of cytochrome p450 reductase in osteoprogenitor cells affects long bone and skull development in mice recapitulating Antley-Bixler syndrome: role of a redox enzyme in development. PLoS ONE 8, e75638. PubMed PMC

Wang M., Roberts D. L., Paschke R., Shea T. M., Masters B. S., and Kim J. J. (1997) Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc. Natl. Acad. Sci. U.S.A. 94, 8411–8416 PubMed PMC

Adachi M., Tachibana K., Asakura Y., Yamamoto T., Hanaki K., and Oka A. (2004) Compound heterozygous mutations of cytochrome P450 oxidoreductase gene (POR) in two patients with Antley-Bixler syndrome. Am. J. Med. Genet. A 128, 333–339 PubMed

Huang N., Pandey A. V., Agrawal V., Reardon W., Lapunzina P. D., Mowat D., Jabs E. W., Van Vliet G., Sack J., Flück C. E., and Miller W. L. (2005) Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis (see comment.). Am. J. Hum. Genet. 76, 729–749 PubMed PMC

Fukami M., Horikawa R., Nagai T., Tanaka T., Naiki Y., Sato N., Okuyama T., Nakai H., Soneda S., Tachibana K., Matsuo N., Sato S., Homma K., Nishimura G., Hasegawa T., and Ogata T. (2005) Cytochrome P450 oxidoreductase gene mutations and Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis: molecular and clinical studies in 10 patients. J. Clin. Endocrinol. Metab. 90, 414–426 PubMed

Adachi M., Asakura Y., Matsuo M., Yamamoto T., Hanaki K., and Arlt W. (2006) POR R457H is a global founder mutation causing Antley-Bixler syndrome with autosomal recessive trait. Am. J. Med. Genet. Part A 140, 633–635 PubMed

Huang N., Agrawal V., Giacomini K. M., and Miller W. L. (2008) Genetics of P450 oxidoreductase: sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations. Proc. Natl. Acad. Sci. U.S.A. 105, 1733–1738 PubMed PMC

Flück C. E., Mullis P. E., and Pandey A. V. (2010) Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism. Biochem. Biophys. Res. Commun. 401, 149–153 PubMed

Tomková M., Marohnic C. C., Gurwitz D., Seda O., Masters B. S., and Martásek P. (2012) Identification of six novel P450 oxidoreductase missense variants in Ashkenazi and Moroccan Jewish populations. Pharmacogenomics 13, 543–554 PubMed PMC

Tomková M., Panda S. P., Šeda O., Baxová A., Hůlková M., Siler Masters B. S., and Martásek P. (2015) Genetic variations in NADPH-CYP450 oxidoreductase in a Czech Slavic cohort. Pharmacogenomics 16, 205–215 PubMed PMC

Krone N., Reisch N., Idkowiak J., Dhir V., Ivison H. E., Hughes B. A., Rose I. T., O'Neil D. M., Vijzelaar R., Smith M. J., MacDonald F., Cole T. R., Adolphs N., Barton J. S., Blair E. M., et al. (2012) Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency. J. Clin. Endocrinol. Metab. 97, E257–E267 PubMed PMC

Marohnic C. C., Huber Iii W. J., Patrick Connick J., Reed J. R., McCammon K., Panda S. P., Martásek P., Backes W. L., and Masters B. S. (2011) Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization. Arch. Biochem. Biophys. 513, 42–50 PubMed PMC

Moutinho D., Marohnic C. C., Panda S. P., Rueff J., Masters B. S., and Kranendonk M. (2012) Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system. Drug Metab. Dispos. 40, 754–760 PubMed PMC

Jin Y., Chen M., Penning T. M., and Miller W. L. (2015) Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency. Biochem. J. 468, 25–31 PubMed PMC

Marohnic C. C., Panda S. P., Martásek P., and Masters B. S. (2006) Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase. J. Biol. Chem. 281, 35975–35982 PubMed

Marohnic C. C., Panda S. P., McCammon K., Rueff J., Masters B. S., and Kranendonk M. (2010) Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect. Drug Metab. Dispos. 38, 332–340 PubMed PMC

Xia C., Panda S. P., Marohnic C. C., Martásek P., Masters B. S., Kim J. J. (2011) Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency. Proc. Natl. Acad. Sci. U.S.A. 108, 13486–13491 PubMed PMC

Gutierrez A., Lian L. Y., Wolf C. R., Scrutton N. S., and Roberts G. C. (2001) Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains. Biochemistry 40, 1964–1975 PubMed

Fontana A., de Laureto P. P., Spolaore B., Frare E., Picotti P., and Zambonin M. (2004) Probing protein structure by limited proteolysis. Acta Biochim. Polonica Engl. Ed. 51, 299–322 PubMed

Heiring C., and Muller Y. A. (2001) Folding screening assayed by proteolysis: application to various cystine deletion mutants of vascular endothelial growth factor. Protein Eng. 14, 183–188 PubMed

Arnold U., Köditz J., Markert Y., and Ulbrich-Hofmann R. (2005) Local fluctuations versus global unfolding of proteins investigated by limited proteolysis. Biocatalysis and Biotransformation 23, 159–167

Royo M., Fitzpatrick P. F., and Daubner S. C. (2005) Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity. Arch Biochem. Biophys. 434, 266–274 PubMed

Hamdane D., Xia C., Im S. C., Zhang H., Kim J. J., and Waskell L. (2009) Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. J. Biol. Chem. 284, 11374–11384 PubMed PMC

Aigrain L., Pompon D., Moréra S., and Truan G. (2009) Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase. EMBO Rep. 10, 742–747 PubMed PMC

Pudney C. R., Khara B., Johannissen L. O., and Scrutton N. S. (2011) Coupled motions direct electrons along human microsomal P450 Chains. PLoS Biol. 9, e1001222. PubMed PMC

Kmoch S., Zeman J., Hrebícek M., Ryba L., Kristensen M. J., and Gregersen N. (1995) Riboflavin-responsive epilepsy in a patient with SER209 variant form of short-chain acyl-CoA dehydrogenase. J. Inherit. Metab. Dis. 18, 227–229 PubMed

Vergani L., Barile M., Angelini C., Burlina A. B., Nijtmans L., Freda M. P., Brizio C., Zerbetto E., and Dabbeni-Sala F. (1999) Riboflavin therapy. Biochemical heterogeneity in two adult lipid storage myopathies. Brain 122, 2401–2411 PubMed

Nicolo C., Flück C. E., Mullis P. E., and Pandey A. V. (2010) Restoration of mutant cytochrome P450 reductase activity by external flavin. Mol. Cell. Endocrinol. 321, 245–252 PubMed

Pallan P. S., Wang C., Lei L., Yoshimoto F. K., Auchus R. J., Waterman M. R., Guengerich F. P., and Egli M. (2015) Human cytochrome P450 21A2, the major steroid 21-hydroxylase: structure of the enzyme.progesterone substrate complex and rate-limiting C-H bond cleavage. J. Biol. Chem. 290, 13128–13143 PubMed PMC

Huber W. J. 3rd., Marohnic C. C., Peters M., Alam J., Reed J. R., Masters B. S., and Backes W. L. (2009) Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system. Drug Metab. Dispos. 37, 857–864 PubMed PMC

Ghosh D., Griswold J., Erman M., and Pangborn W. (2009) Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature 457, 219–223 PubMed PMC

Ghosh D., Griswold J., Erman M., and Pangborn W. (2010) X-ray structure of human aromatase reveals an androgen-specific active site. J. Steroid Biochem. Mol. Biol. 118, 197–202 PubMed PMC

Wang Y.-H., Tee M. K., and Miller W. L. (2010) Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase A. Endocrinology 151, 1677–1684 PubMed PMC

Yoshimoto F. K., Zhou Y., Peng H.-M., Stidd D., Yoshimoto J. A., Sharma K. K., Matthew S., and Auchus R. J. (2012) Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates. Biochemistry 51, 7064–7077 PubMed PMC

Yoshimoto F. K., Peng H.-M., Zhang H., Anderson S. M., and Auchus R. J. (2014) Epoxidation activities of human cytochromes P450c17 and P450c21. Biochemistry 53, 7531–7540 PubMed PMC

Mui B., and Hope M. J. (1993) Formation of Large Unilamellar Vesicles by Extrusion, in Liposome Technology (Gregoriadis G., ed) 3rd Ed., pp. 55–66, CRC Press, Boca Raton, FL

McPherson A. (1999) Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Stura E. A., and Wilson I. A. (1990) Analytical and production seeding techniques. Methods 1, 38–49

Otwinowski Z., and Minor W. (1997) Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 PubMed

Vagin A., and Teplyakov A. (2010) Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 PubMed

Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 PubMed PMC

Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S., Read R. J., Rice L. M., Simonson T., and Warren G. L. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 PubMed

Emsley P., and Cowtan K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 PubMed

McLaughlin L. A., Dickmann L. J., Wolf C. R., and Henderson C. J. (2008) Functional expression and comparative characterization of nine murine cytochromes P450 by fluorescent inhibition screening. Drug Metab. Dispos. 36, 1322–1331 PubMed

Makaji E., Trambitas C. S., Shen P., Holloway A. C., and Crankshaw D. J. (2010) Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms. Toxicol. Sci. 113, 293–304 PubMed

Lala P., Higashiyama T., Erman M., Griswold J., Wagner T., Osawa Y., and Ghosh D. (2004) Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex. J. Steroid Biochem. Mol. Biol. 88, 235–245 PubMed

Duarte M. P., Palma B. B., Laires A., Oliveira J. S., Rueff J., and Kranendonk M. (2005) Escherichia coli BTC, a human cytochrome P450 competent tester strain with a high sensitivity towards alkylating agents: involvement of alkyltransferases in the repair of DNA damage induced by aromatic amines. Mutagenesis 20, 199–208 PubMed

Kranendonk M., Marohnic C. C., Panda S. P., Duarte M. P., Oliveira J. S., Masters B. S., and Rueff J. (2008) Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase. Arch. Biochem. Biophys. 475, 93–99 PubMed PMC

Fisher C. W., Caudle D. L., Martin-Wixtrom C., Quattrochi L. C., Tukey R. H., Waterman M. R., and Estabrook R. W. (1992) High-level expression of functional human cytochrome P450 1A2 in Escherichia coli. FASEB J. 6, 759–764 PubMed

Kranendonk M., Mesquita P., Laires A., Vermeulen N. P., and Rueff J. (1998) Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens. Mutagenesis 13, 263–269 PubMed

Zobrazit více v PubMed

PDB
3QE2, 5EMN, 5FA6