title: Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.
abstract: Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and -300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products.
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|VILLUM Research Center for Plant Plasticity, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|Center for Synthetic Biology 'bioSYNergy', Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.
authors.forename: Krutika
authors.initials: K
authors.lastname: Bavishi
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|VILLUM Research Center for Plant Plasticity, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|Center for Synthetic Biology 'bioSYNergy', Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|Joint BioEnergy Institute, Feedstocks Division, Emeryville, CA 94608, USA.
authors.forename: Tomas
authors.initials: T
authors.lastname: Laursen
authors.affiliation: Center for Synthetic Biology 'bioSYNergy', Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|Bio-Nanotechnology Laboratory, Department of Chemistry &Nano-Science Center, Universitetparken 5, DK-2100, University of Copenhagen, Denmark.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Martinez
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|VILLUM Research Center for Plant Plasticity, Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.|Center for Synthetic Biology 'bioSYNergy', Thorvaldsensvej 40, DK-1871 Frederiksberg C, University of Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Bio-Nanotechnology Laboratory, Department of Chemistry &Nano-Science Center, Universitetparken 5, DK-2100, University of Copenhagen, Denmark.
authors.forename: Eduardo Antonio
authors.initials: EA
authors.lastname: Della Pia
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D049329:Nanostructures; D010940:Plant Proteins; D045868:Sorghum / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D009251:NADPH-Ferrihemoprotein Reductase