Reconstitution of human cytochrome P450 activity using a Leishmania cell-free protein expression system

Cytochrome P450 enzymes (P450s) are ubiquitous in drug metabolism and natural product biosynthesis. Studying eukaryotic P450s has been limited by their dependence on membrane association and requirement for partner reductases. Here, we demonstrate cell-free synthesis and assay of human P450s 3A4 and 2D6 using Leishmania tarentolae translational extract. These P450s were co-expressed with various NADPH-cytochrome P450 reductases (CPRs), and activity was assayed directly using unpurified reactions. P450s 3A4 and 2D6 showed distinct preferences in reductase coupling: P450 3A4 activity was greatest when coupled to the human CPR, whereas P450 2D6 performed better when co-expressed with CPRs from Arabidopsis thaliana . Inhibition assays with chloramphenicol, terbinafine, and erythromycin yielded results consistent with known P450-drug interactions. We conclude that Leishmania -based cell-free protein synthesis resolves previous challenges in eukaryotic P450 expression, allowing for rapid and convenient functional studies of unmodified eukaryotic P450 systems, offering a practical tool for drug metabolism studies and biocatalyst discovery.