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.
Highlights
Cytochromes P450 have been aptly nicknamed “nature’s blow torches” owing to their ability to catalyze an impressive diversity of complex and chemically unfavoured reactions with high specificity and stereo-selectivity
Purified CYP79A1, CYP71E1 and P450 oxidoreductase (POR) were reconstituted in nanodiscs containing a mixture of phospholipids (DLPC, DLPG, and NBD-PC) and assembled using the membrane scaffold proteins MSP1E3D1
Since the first direct electrochemistry report of P450cam by Hill et al in 199650, studies of P450 enzymes have been restricted to enzymes of microbial and human origin with focus on their fundamental application in xenobiotic degradation and drug metabolism[38]
Summary
CYP79A1 and CYP71E1 with a 6x his-tag at the C-termini and cloned into the pYeDP60 vector were expressed in S. cerevisiae BY4741 strains and purified by Ni-NTA affinity chromatography. The activity of nanodiscs containing CYP79A1/ CYP71E1 may seem reduced when compared to the detergent solubilized version This is due to utilization of non-native soluble electron donors Fd/FNR. The detergent solubilized and nanodisc reconstituted POR displayed turnover rates of 2063 and 3000 min−1, respectively, which are comparable to previously published values[26] (Fig. 4C). The redox potential obtained from direct electrochemistry of immobilized non-symbiotic plant hemoglobin class II from Beta vulgaris[43] in the absence of any electron mediators was reported to be 171 mV (vs NHE) This is a significant anodic shift as compared to other hemoglobins (−1 00 to −1 26 mV)[44,45] indicating the midpoint potentials of heme proteins can vary considerably. These strategies attempted to mimic the natural electron flux via a cognate redox protein and have been largely successful
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