NADPH-cytochrome P450 reductase (CPR) catalyzes the transfer of reducing equivalents from NADPH to cytochromes P450 and is an essential component of the microsomal cytochrome P450 monooxygenase system. The system carries out oxidative metabolism of various drugs, xenobiotics, and a number of endogenous substrates, including steroids, lipids, and prostaglandins. CPR and nitric oxide synthase are the only two mammalian enzymes known to contain both flavin prosthetic groups, FMN and FAD, and an NADPH binding site. Despite the intensive studies over the last three decades to elucidate the mechanism, structure, and genetic regulation of CPR, large gaps in our understanding still exist. Detailed structural information from high resolution X-ray analysis will enable us to relate biochemical functions to the structure of the protein and to define the catalytic mechanism. We have obtained X-ray quality crystals of rat liver CPR that has been cloned and expressed in E. coli and solubilized by limited trypsinolysis. We have also obtained two heavy atom derivatives. We propose to determine the three-dimensional structure of CPR at high resolution in its native and NADP+/NADPH bound farms. Structures of the site-specific mutants designed to probe its catalytic mechanism and regulation will also be determined. Initiation of the crystallographic studies on the intact CPR is also proposed to study the interactions between CPR and its physiological electron acceptor, cytochromes P450.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052682-04
Application #
2883026
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1996-03-01
Project End
2002-02-28
Budget Start
1999-03-01
Budget End
2002-02-28
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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Hamdane, Djemel; Xia, Chuanwu; Im, Sang-Choul et al. (2009) Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. J Biol Chem 284:11374-84
Sarapusit, Songklod; Xia, Chuanwu; Misra, Ila et al. (2008) NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability. Arch Biochem Biophys 477:53-9
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