title: Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase.
abstract: A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine. Transformation of Escherichia coli (DH5 alpha) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein). A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein. In the presence of NADPH the purified fusion protein catalyzes the 17 alpha-hydroxylation of progesterone and pregnenolone as well as the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone. The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5. Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17 alpha-hydroxy-progesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5. Use of 5 alpha-pregnan steroids as substrates show the importance of the 3 beta-hydroxyl group for cytochrome b5 stimulated 17,20-lyase activity. Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235.
authors.forename: M S
authors.initials: MS
authors.lastname: Shet
authors.forename: C W
authors.initials: CW
authors.lastname: Fisher
authors.forename: M P
authors.initials: MP
authors.lastname: Arlotto
authors.forename: C H
authors.initials: CH
authors.lastname: Shackleton
authors.forename: P L
authors.initials: PL
authors.lastname: Holmans
authors.forename: C A
authors.initials: CA
authors.lastname: Martin-Wixtrom
authors.forename: Y
authors.initials: Y
authors.lastname: Saeki
authors.forename: R W
authors.initials: RW
authors.lastname: Estabrook
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015183:Restriction Mapping; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D009249:NADP; D015786:Cytochromes b5; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase