Expression and characterization of cytochrome P4504A7 in E.coli

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

Research output: Contribution to journalArticlepeer-review

Abstract

The cytochromes P450 (P450) constitute a family of hemeproteins that serve as monooxygenases for exogenous and endogenous substrates. The P4504A subfamily, consisting of eicosanoid and fatty acid hydroxylases, plays a role in the metabolism of arachidonic acid in lung, liver and kidney. This metabolism of arachidonic acid produces 20-hydroxyeicosatetraenoic acid (20-HETE), shown to be a potent vasoconstrictor and, consequently, could be involved in hemodynamic regulation (Sawamura et al., 1993). The P4504A family members from rabbit, 4A4, 4A5, 4A6, and 4A7, are 85% homologous in amino acid sequence 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 (20nmoles/liter) as a chimera with the first ten amino acids of bovine P45017a. Kinetic characterization determined that the primary substrate, lauric acid, was metabolized at a turnover number of 20min1, with a Km of 1.6nM. Characterization of other substrates will be presented.

Original languageEnglish (US)
Pages (from-to)A805
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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