Combined surface display of Cytochrome P450 1A2 and its reductase on Escherichia coli

Quehl P, Jose J

Abstract

Human cytochrome P450 monooxygenases (CYPs) play a prominent role in drug metabolism as these enzymes are involved in the breakdown of literally every drug. They catalyze a variety of oxidations of a broad range of substrates and have a major impact on bioavailability and drug-drug interactions. CYPs obtain the electrons from the NADPH-dependent cytochrome P450 reductase (CPR). However, recombinant expression of both enzymes in bacteria is often not feasible and they exhibit poor stability after purification. Moreover, the membrane associated proteins CPR and CYPs require membrane surroundings for activity. CYPs alone can be displayed functionally active on the surface of Escherichia coli with externally added CPR. Here, we tested the co-expression of CYP1A2 and its NADPH dependent Cytochrome P450 reductase (CPR) on the E.coli outer membrane. 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 domain. Surface presentation was confirmed by FACS analysis and protease accessibility tests. Both CYP1A2 and CPR bind their cofactors and the surface displayed NADPH-dependent cytochrome P450 reductase is able to react with Cytochrome C.