Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers.
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.
Duan
Hui
H
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Civjan
Natanya R
NR
Sligar
Stephen G
SG
Schuler
Mary A
MA
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism*; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006793:Houseflies / Q000201:enzymology; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008567:Membranes, Artificial; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036103:Nanotechnology / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D013379:Substrate Specificity; D050564:Trans-Cinnamate 4-Monooxygenase
D001672:Biocompatible Materials; D002934:Cinnamates; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D011994:Recombinant Proteins; C029010:cinnamic acid; D002245:Carbon Dioxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase