Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 424, Issue 2, Pages 141-153Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2004.02.010
Keywords
cytochrome P450 monooxygenases; Arabidopsis P450s; nanoscale lipid bilayers (Nanodiscs); P450 reductases; heterologous expression systems
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Funding
- NIGMS NIH HHS [R01GM33775] Funding Source: Medline
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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. (C) 2004 Elsevier Inc. All rights reserved.
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