Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers

Hui Duan, Natanya R. Civjan, Stephen G. Sligar, Mary A. Schuler

Research output: Contribution to journalArticlepeer-review

Abstract

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His 6-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.

Original languageEnglish (US)
Pages (from-to)141-153
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume424
Issue number2
DOIs
StatePublished - Apr 15 2004

Keywords

  • Arabidopsis P450s
  • Cytochrome P450 monooxygenases
  • Heterologous expression systems
  • Nanoscale lipid bilayers (Nanodiscs)
  • P450 reductases

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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