Expression and characterization of cytochrome P4504A7 in E. coli

P. A. Loughran, L. J. Roman, Bettie S Masters

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The cytochromes P450 (P450) constitute a family of hemeproteins that serve as monooxygenases for exogenous and endogenous substrates. Certain members of the P4504A subfamily, which consists of eicosanoid and fatty acid hydroxylases, play a role in the metabolism of arachidonic acid in lung, liver and/or kidney. This metabolism of arachidonic acid produces 20hydroxyeicosatetraenoic acid (20-HETE), shown to be a potent vasoconstrictor, thus implicating these P450s involved in hemodynamic regulation (Sawamura et al., 1993). The P4504A family members from rabbit, 4A4, 4A5, 4A6, and 4A7, are 85% homologous at the amino acid level but have been shown to exhibit differing substrate specificities (Roman et al., 1993). To determine the structural and functional characteristics of the P4504A7 substrate binding/recognition site, production of large amounts of enzyme in an E.coli expression was accomplished (6-8 nmoles P450/liter of cells). Having optimized the assay conditions (NADPH-cytochrome P450 reductase, lipid, cytochrome b,), metabolism of lauric and arachidonic acid in the presence of cytochrome b, exhibited a turnover number of 130 min-1 and 74 min-1, with Km values of 27 μM and 74.5 μM, respectively. In the absence of cytochrome b5 lauric acid metabolism exhibited a turnover number of 28 min-1 and a Km of 3.9 μM. Arachidonic acid metabolism in the absence of cytochrome b5 is not detectable.

Original languageEnglish (US)
Pages (from-to)A1421
JournalFASEB Journal
Issue number8
StatePublished - Dec 1 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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