Molecular Cloning, Expression and Characterization of an Endogenous Human Cytochrome P450 Arachidonic Acid Epoxygenase Isoform

Abstract

A cDNA containing an open reading frame coding for a human cytochrome P450 arachidonic acid epoxygenase was isolated from a male human kidney cDNA library. Sequence analysis showed that, with few exceptions, this cDNA was nearly identical to the published sequence for human liver Cyp 2C8 (S. T. Okino et al., 1987, J. Biol. Chem. 262, 16072-16079) and encoded a polypeptide of 490 amino acids. Nucleic acid hybridization indicated that: (a) Cyp 2C8 and 2C10 were expressed at comparable levels in the human liver and (b) compared to Cyp 2C10, the steady state concentrations of Cyp 2C8 transcripts in the human kidney were substantially lower. The kidney 2C8 cDNA was cloned into a pBlue BacIII vector, expressed using a baculovirus/ Sf9 insect cell system, and the recombinant Cyp 2C8 protein was purified by a combination of hydrophobic and hydroxylapatite chromatography. Purified recombinant Cyp 2C8 and 2C10 were reconstituted in the presence of NADPH and NADPH-cytochrome P450 reductase and shown to metabolize arachidonic via olefin epoxidation with both proteins generating, almost exclusively, epoxygenase-derived products (94 and 90% of total products, respectively). Catalytic turnover (1.05 and 0.75 nmol of product/nmol of hemoprotein/min at 30°C for Cyp 2C8 and 2C10, respectively) was inhibited by the addition of purified cytochrome b 5. Metabolism by recombinant 2C8 was both regio- and enantioselective for 11( R), 12( S)- and 14( R), 15( S)-epoxyeicosatrienoic acids (82% optical purity, each). Compared to Cyp 2C8, arachidonic acid epoxidation by Cyp 2C10 was less regio- and stereoselective and generated mixtures of 8( S), 9( R)-, 11( S), 12( R)-, and 14( R), 15( S)-epoxyeicosatrienoic acids (with optical purities of 66, 69, 63%, respectively). Importantly, recombinant Cyp 2C8 and 2C10 epoxidized the arachidonic acid 11, 12-olefin with opposite enantiofacial selectivities. Only for Cyp 2C8 did the chirality of the products match that of the enantiomers present, in vivo in human kidney cortex (A. Karara et al. 1990, FEBS Lett. 268, 227-230). Hence, we propose that Cyp 2C8 is one of the human cytochrome P450 isoforms responsible for the metabolism of endogenous arachidonic acid pools.