Using cDNA clones prepared in previous studies in this laboratory in the modified Okayama-Berg cloning vector, it is proposed to construct and express chimeric P-450 steroid hyroxylases in COS 1 cells of monkey kidney origin in order to examine the role of specific P-450 sequences in steroid hydroxylase activities. In particular, chimeric proteins will be constructed between P-45017 alpha and P450C21 and between P-45017 alpha and P-450scc. The P-450 domains to be examined in these studies include the Ozols tridecapeptide and the heme-binding region. It will be tested whether either of these regions of P-450C21 can impart 21- hydroxylase activity to P-45017 alpha. In addition it will be tested whether either of these regions of P-450scc can impart cholesterol side chain cleavage activity to P-45017 alpha and whether either of these regions of P-45017 alpha can impart 17 alpha-hydroxylase/17, 20-lyase activity to P-450scc. These chimeric proteins will be prepared from their specific cDNA inserts utilizing the two-primer mutagenesis procedure of Zoller and Smith. The resultant chimeric cDNA molecules will be expressed in COS 1 cells using the same procedure previously utilized by this laboratory to express P-45017 alpha, P-450scc and adrenodoxin cDNAs. Also it is proposed to carry out site-specific mutagenesis of specific amino acid residues of anrenodoxin by this procedure. Finally, the two-primer mutagenesis procedure will be utilized to investigate the amino acid sequence requirements for insertion of P-45017 alpha into the endoplasmic reticulum and P- 450scc and adrenodoxin into their correct submitochondrial compartments. These studies represent the first investiggation of expression and structure-function relationships of P-450 and adrenodoxin using mammaliam cells. The results of these studies will provide information on specific steroid hydroxylase structure- function relationships as well as general aspects of P-450 structure vs. function. In addtion they will provide new insights into the biogenesis of functional steroid hydroxylase complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037942-05
Application #
3293808
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1987-04-01
Project End
1992-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Oyama, Tsunehiro; Kagawa, Norio; Sugio, Kenji et al. (2009) Expression of aromatase CYP19 and its relationship with parameters in NSCLC. Front Biosci (Landmark Ed) 14:2285-92
Miller, Todd W; Shin, Incheol; Kagawa, Norio et al. (2008) Aromatase is phosphorylated in situ at serine-118. J Steroid Biochem Mol Biol 112:95-101
Tosha, Takehiko; Kagawa, Norio; Arase, Miharu et al. (2008) Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy. J Biol Chem 283:3708-17
Zollner, Andy; Kagawa, Norio; Waterman, Michael R et al. (2008) Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli. FEBS J 275:799-810
Podust, Larissa M; von Kries, Jens P; Eddine, Ali Nasser et al. (2007) Small-molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by X-ray crystallography. Antimicrob Agents Chemother 51:3915-23
Lamb, David C; Guengerich, F Peter; Kelly, Steven L et al. (2006) Exploiting Streptomyces coelicolor A3(2) P450s as a model for application in drug discovery. Expert Opin Drug Metab Toxicol 2:27-40
Lamb, David C; Kim, Youngchang; Yermalitskaya, Liudmila V et al. (2006) A second FMN binding site in yeast NADPH-cytochrome P450 reductase suggests a mechanism of electron transfer by diflavin reductases. Structure 14:51-61
Sherman, David H; Li, Shengying; Yermalitskaya, Liudmila V et al. (2006) The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J Biol Chem 281:26289-97
Tosha, Takehiko; Kagawa, Norio; Ohta, Takehiro et al. (2006) Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps. Biochemistry 45:5631-40
Arase, Miharu; Waterman, Michael R; Kagawa, Norio (2006) Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli. Biochem Biophys Res Commun 344:400-5

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