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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Baculoviridae / enzymology
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Baculoviridae / genetics
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Biocompatible Materials / chemistry
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Biocompatible Materials / metabolism*
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Carbon Dioxide / chemistry
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Carbon Dioxide / metabolism
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Cells, Cultured
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Cinnamates / chemistry
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Cinnamates / metabolism
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Cytochrome P-450 Enzyme System / chemistry
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism*
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Enzyme Activation
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Houseflies / enzymology
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Lipid Bilayers / chemistry
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Lipid Bilayers / metabolism*
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Membranes, Artificial
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Microsomes / chemistry
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Microsomes / enzymology*
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Mixed Function Oxygenases / chemistry
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Mixed Function Oxygenases / genetics
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Mixed Function Oxygenases / metabolism*
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Molecular Weight
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NADPH-Ferrihemoprotein Reductase / chemistry
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NADPH-Ferrihemoprotein Reductase / genetics
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NADPH-Ferrihemoprotein Reductase / metabolism*
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Nanotechnology / methods*
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Protein Engineering / methods*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Solubility
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Substrate Specificity
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Trans-Cinnamate 4-Monooxygenase
Substances
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Biocompatible Materials
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Cinnamates
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Lipid Bilayers
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Membranes, Artificial
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Recombinant Proteins
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cinnamic acid
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Carbon Dioxide
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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Trans-Cinnamate 4-Monooxygenase
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NADPH-Ferrihemoprotein Reductase