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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.

SUBMITTER: Bavishi K

PROVIDER: S-EPMC4937447 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

Bavishi Krutika K Laursen Tomas T Martinez Karen L KL Møller Birger Lindberg BL Della Pia Eduardo Antonio EA

Scientific reports 20160708

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 immo ...[more]

PMID: 27386958

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Project description:The influence of the side chains and positioning of the carboxy-terminal residues of NADPH-cytochrome P450 oxidoreductase (CYPOR) on catalytic activity, structure of the carboxy terminus, and interaction with cofactors has been investigated. A tandem deletion of residues Asp675 and Val676, that was expected to shift the position of the functionally important Trp677, resulted in higher cytochrome c reductase activity than that expected from previous studies on the importance of Asp675 and Trp677 in catalysis. Crystallographic determination of the structure of this variant revealed two conformations of the carboxy terminus. In one conformation (Mol A), the last α-helix is partially unwound, resulting in repositioning of all subsequent residues in β-strand 21, from Arg671 to Leu674 (corresponding to Ser673 and Val676 in the wild type structure). This results in the two C-terminal residues, Trp677 and Ser678, being maintained in their wild type positions, with the indole ring of Trp677 stacked against the isoalloxazine ring of FAD as seen in the wild type structure, and Ser673 occupying a similar position to the catalytic residue, Asp675. The other, more disordered conformation is a mixture of the Mol A conformation and one in which the last α-helix is not unwound and the nicotinamide ring is in one of two conformations, out towards the protein surface as observed in the wild type structure (1AMO), or stacked against the flavin ring, similar to that seen in the W677X structure that lacks Trp677 and Ser678 (1JA0). Further kinetic analysis on additional variants showed deletion or substitution of alanine or glycine for Trp677 in conjunction with deletion of Ser678 produced alterations in interactions of CYPOR with NADP+, 2'5'-ADP, and 2'-AMP, as well as the pH dependence of cytochrome c reductase activity. We postulate that deletion of bulky residues at the carboxy terminus permits increased mobility leading to decreased affinity for the 2'5'-ADP and 2'-AMP moieties of NADP+ and subsequent domain movement.

Dataset Information

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

Publications

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

Similar Datasets

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