Combined display of cytochrome P450 reductase and CYP 1A2 on the surface of Escherichia coli.

Quehl P, Riemer J, Jose J

Abstract

Administering a drug requires a profound knowledge about its metabolism in the human body. Cytochrome P450 monooxygenases (CYPs) take part in the breakdown of almost every drug catalyzing a variety of oxidations of a broad range of substrates1. Understanding their enzymatic behavior is crucial for drug development as CYP activity affects bioavailability and drug-drug interactions. They obtain the electrons from the NADPH-dependent cytochrome P450 reductase (CPR)2. However, recombinant expression of both enzymes is often not feasible in bacteria since they require membrane surroundings for activity. Furthermore, the purified enzymes are poorly stable. It has been shown that CYPs alone can be displayed functionally active on the outer membrane of Escherichia coli with externally added CPR3. Here, we tested the co-expression of human CPR with CYP 1A2 on E. coli. Surface display is facilitated by usage of the autotransporter secretion pathway for which the enzyme of interest is combined with an N-terminal signal peptide, a C-terminal linker and a beta-barrel domain3,4. Surface exposure was confirmed by FACS analysis and test of protease accessibility. The surface displayed CPR is able to react with cytochrome C5. Fluorescence spectra indicate that both CYP1A2 and CPR fusion proteins bind their cofactors. The functional interaction of CYP1A2 with the CPR is currently under investigation by testing the enzymatic activity towards the substrates 7-ethoxyresorufin and phenacetin.