Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs

Yelena V. Grinkova, Ilia G. Denisov, Stephen G. Sligar

Research output: Contribution to journalArticle

Abstract

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37°C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug-drug interaction studies.

Original languageEnglish (US)
Pages (from-to)194-198
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume398
Issue number2
DOIs
StatePublished - Jul 2010

Keywords

  • Cytochrome P450
  • Nanodisc
  • P450 reductase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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