Cytochrome P450 bienzymes assembled on Au/chitosan/reduced graphene oxide nanosheets for electrochemically-driven drug cascade metabolism

Inspired by the high enzymatic reaction efficiency of the natural multi-enzyme complexes, construction of artificial multi-enzyme complexes for mimicking of the natural metabolic pathways in vitro has attracted significant interest from enzyme engineers. Herein, we have successfully assembled a cytochrome P450 (CYP) bienzyme complex on the Au nanoparticle/chitosan/reduced graphene oxide nanocomposite sheets (Au/CS/RGO) to explore the drug cascade metabolism using an electrochemically-driven approach. When model bienzymes, CYP1A2 and CYP3A4 isozymes, were sequentially assembled on Au/CS/RGO, one pair of well-defined redox peaks at -0.531 and -0.474 V (vs. SCE) was observed due to the overlap of the redox peaks of CYP1A2 and CYP3A4, confirming a good electrochemical activity of the CYP bienzyme complex. With an electrochemically-driven approach, the CYP bienzyme complex displayed synergic functions, whereby the intermediate of 2-oxo-clopidogrel generated from target substrate clopidogrel by CYP1A2 could be promptly converted into the final metabolite of clopidogrel carboxylic acid by CYP3A4. This sequential conversion could be demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Furthermore, the as-constructed CYP bienzyme complex could be also used in-situ to monitor the clopidogrel concentration with sensitivity of 143.4 μA mM−1 and detection limit of 0.63 μM. Therefore, the successful construction of the CYP bienzyme complex would offer a platform for studying the cascade enzymatic reaction, and provide a potential application in efficient biosensors for toxicity analysis and bioreactors for chemical synthesis.