Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

Rahul Yadav,Emily E Scott

doi:10.1074/jbc.ra118.005417

Rahul Yadav, Emily E Scott

Open Access

https://doi.org/10.1074/jbc.ra118.005417

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Abstract

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

Highlights

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases
The ␦-aminolevulinic acid is readily taken up by E. coli, where it is used in heme synthesis by bacterial enzymes, and the heme is endogenously inserted into a human P450 polypeptide to produce enzymatically active protein
Despite the significant potential contributions in probing human P450 function and structure, we have found no reports of successful generation of metal-substituted membrane cytochrome P450 enzymes, which even in their native FePPIX state are often quasi-stable

Summary

Results and discussion

Initial trials reconfirmed that protoporphyrin extraction and reconstitution was not a feasible approach for substituting the prosthetic group in membrane P450 enzymes due to excessive precipitation of apo-P450 polypeptide and poor reincorporation of even the native FePPIX. NMR permits evaluation of the entire CYP17A1 structure under different conditions, even if the spectrum is crowded by Ͼ500 amino acid resonances For this purpose, CYP17A1 was generated in the presence of the abiraterone inhibitor with either exogenous PPIX supplementation or endogenous PPIX biosynthesis while substituting 15N-ammonium chloride for the unlabeled ammonium chloride in the minimal medium. When FePPIX is exogenously supplemented, CYP3A4, CYP21A2, and CYP17A1 can be generated with normal spectroscopic features and often identical steady-state kinetic constants for their respective substrate oxidations Using this methodology, other exogenous metal protoporphyrins could be readily incorporated into CYP17A1, often without significant perturbation in the overall protein structure as evaluated by 2D NMR. In addition to the biophysical application in NMR discussed other structural and mechanistic studies can profit from having control of the metal center at the heart of membrane P450 catalysis driving human drug metabolism and key endogenous and disease-related pathways such as steroidogenesis

Plasmid constructs

Protein expression and purification

Functional assays

NMR spectroscopy