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)

Fah1p, a Saccharomyces cerevisiae Cytochromeb 5 Fusion Protein, and ItsArabidopsis thaliana Homolog That Lacks the Cytochromeb 5 Domain Both Function in the α-Hydroxylation of Sphingolipid-associated Very Long Chain Fatty Acids

“…To induce expression of the recombinant protein, 2 ml of an overnight culture were used to inoculate 100 ml of LB (200 g/ml ampicillin). The cultures were then grown with shaking (250 rpm) at 37°C for 2 h. Thirty minutes before induction with 1 mM isopropyl-1-thio-␤-D-galactopyranoside, a 75 g/liter concentration of the heme precursor 5-aminolevulinic acid was added (27), and the cells were then grown for 20 h at the reduced temperature of 30°C (28). The cells were harvested by centrifugation at 5000 ϫ g for 5 min and washed once with 100 mM Tris-HCl, pH 8, 1 mM phenylmethylsulfonyl fluoride.…”

Characterization of a New Hemoprotein in the YeastSaccharomyces cerevisiae

“…coli cells transformed with this plasmid were grown in liquid culture supplemented with the heme precursor 5-aminolevulinic acid to increase de novo heme synthesis (40). After induction with isopropyl ␤-D-thiogalactopyranoside, a prominent protein band of the expected 46 kDa was visible in bacterial extracts.…”

Nutritional Status Modulates Rat Liver Cytochrome P450 Arachidonic Acid Metabolism

“…Studies using purified and/or recombinant enzymes have demonstrated that multiple P450s can metabolize AA and that the products depend largely on the particular P450 enzyme involved in catalysis. For example, members of the CYP2B and CYP2C subfamilies are primarily AA epoxygenases (Capdevila et al, 1990a;Rifkind et al, 1995); members of the CYP1A, CYP2E, and CYP4A subfamilies are principally -terminal hydroxylases (Capdevila et al, 1990a;Laethem et al, 1993;Nishimoto et al, 1993;Rifkind et al, 1995); and members of the CYP2J subfamily are both epoxygenases and -terminal hydroxylases (Wu et al, 1997). The epoxygenase and hydroxylase reactions are both regioselective and enantioselective, and the reaction asymmetry is P450 enzyme specific (Capdevila et al, 1990a).…”