Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5

Gihan Hosny, Linda J. Roman, Mostafa H. Mostafa, Bettie Sue S. Masters

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (ω- hydroxylation) and, to a lesser extent, at the penultimate carbon [(ω- 1)hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of ~80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate- perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The k(cat) and K(m) values for laurate ω-hydroxylation were 41 min-1 and 8.5 μM, respectively, in the absence of cytochrome b5, and 138 min-1 and 38 μM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; k(cat) and K(m) values were 48 min-1 and 122 μM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.

Original languageEnglish (US)
Pages (from-to)199-206
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume366
Issue number2
DOIs
StatePublished - Jun 15 1999

Keywords

  • 4A5
  • Cytochrome P450
  • Fatty acid hydroxylase
  • Lauric acid
  • P450
  • P4504A5

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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