CYTOCHROME-P450-4A4 - EXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION OF CATALYTIC PROPERTIES

Rabbit lung prostaglandin omega-hydroxylase (P450 4A4) was expressed i n Escherichia coli using the isopropyl beta-D-thiogalactopyranoside (I PTG) inducible expression vector pCWori+, containing the full-length c DNA encoding the P450 4A4. The first seven codons were changed to refl ect 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 r esidue of P450 4A4 was altered (Ser to Ala), while the remaining mutat ions were silent. This strategy was adopted in order to minimize chang es 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 el ectrophoretic homogeneity, and kinetic and spectrophotometric data ind icate that this expressed, purified enzyme is equivalent to the enzyme purified from rabbit lung. The K(m) for PGE1 was determined to be 3.0 muM, 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(ma x) at 450 nm, and the absolute absorbance spectrum of the pyridine hem ochromogen revealed a typical b type heme. To characterize P450 4A4 fu rther, the catalytic activities with prostaglandin E1 (PGE1), arachido nate, 15-hydroxyeicosatetraenoic acid (15-HETE), and palmitate were in vestigated. PGE1 and arachidonate omega-hydroxylation activities were highly dependent on the concentration of NADPH-cytochrome P450 oxidore ductase, with maximal activities being achieved at a 10-20-fold excess of reductase. Interestingly, activities with arachidonate, palmitate, and 15-HETE, but not with PGE1, were found to be highly dependent on the amount of L-alpha-dilauroylphosphatidylcholine (DLPC) in the react ion mixtures. Using optimal amounts of DLPC, initial velocity kinetic experiments were performed. Surprisingly, in spite of structural dissi milarity among these substrates, the K(m) values did not differ signif icantly. Cytochrome b5 had basically no effect on the K(m) values but doubled V(max) values for PGE1, palmitate, and 15-HETE and tripled the V(max) for arachidonate. The V(max) for arachidonate was found to be the highest, 37 pmol min-1 pmol-1, and for PGE1 the lowest, 8.4 pmol m in-1 pmol-1, determined in the presence of cytochrome b5. The V(max)/K (m) values were determined to be 22, 10, 6.1, and 2.8 for arachidonate , palmitate, 15-HETE, and PGE1, respectively, in the presence of cytoc hrome b5. These results demonstrate that P450 4A4 utilizes arachidonat e efficiently and suggest that this enzyme is contributing to the phys iological co-hydroxylation of arachidonate.