title: Cytochrome P450 4A4: expression in Escherichia coli, purification, and characterization of catalytic properties.
abstract: Rabbit lung prostaglandin omega-hydroxylase (P450 4A4) was expressed in Escherichia coli using the isopropyl beta-D-thiogalactopyranoside (IPTG) inducible expression vector pCWori+, containing the full-length cDNA encoding the P450 4A4. The first seven codons were changed to reflect E. coli codon bias [a modification of the method of Barnes et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5597-5601]; only the second residue of P450 4A4 was altered (Ser to Ala), while the remaining mutations were silent. This strategy was adopted in order to minimize changes in the structure of the expressed enzyme. Induction by IPTG of the apoprotein peaked after 6 h, and by including the heme precursor delta-aminolevulinic acid, enzymatic activity peaked 12 h after addition of IPTG. The isolated membrane fraction, free of cell debris, contained 12-15 nmol of P450/L of media. The expressed enzyme was purified to electrophoretic homogeneity, and kinetic and spectrophotometric data indicate that this expressed, purified enzyme is equivalent to the enzyme purified from rabbit lung. The Km for PGE1 was determined to be 3.0 microM, which is the same as that obtained for the enzyme purified from lung [Williams et al. (1984) J. Biol. Chem. 259, 14600-14608]. The CO-reduced difference spectrum of purified P450 4A4 exhibited a lambda max at 450 nm, and the absolute absorbance spectrum of the pyridine hemochromogen revealed a typical b type heme. To characterize P450 4A4 further, the catalytic activities with prostaglandin E1 (PGE1), arachidonate, 15-hydroxyeicosatetraenoic acid (15-HETE), and palmitate were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: M
authors.initials: M
authors.lastname: Nishimoto
authors.forename: J E
authors.initials: JE
authors.lastname: Clark
authors.forename: B S
authors.initials: BS
authors.lastname: Masters
mesh_terms: D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D004247:DNA; D004790:Enzyme Induction; D004926:Escherichia coli; D006900:Hydroxylation; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011817:Rabbits
chemical_list: D007527:Isoenzymes; D010743:Phospholipids; D007544:Isopropyl Thiogalactoside; D000622:Aminolevulinic Acid; D004247:DNA; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C026961:prostaglandin omega hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase