Expression and characterization of cytochrome P450 4A5 in E. coli

Cytochromes P450 constitute a superfamily of hemoproteins that catalyze the oxidative biotransformation of lipophilic substrates. Cytochromes P450 of the 4A family catalyze lo-hydroxylation of a variety of fatty acids, prostaglandins and eicosanoids. Cytochrome P450 4A5 has been successfully expressed in E. coli using the IPTG-inducible expression vector pCW0,., containing the full-length cDNA encoding the histidinetagged P450 4A5, under (ac promoter control. The average yield of enzyme was 80-100 nmol/liter of cells. Purification of 4A5 yielded highly purified enzyme, approximately 50% detergent-free and 50% detergentpurified forms. The detergent-free form was characterized spectroscopically, using EPR and absolute and substrate-perturbed difference optical spectroscopy. EPR spectra showed that the heme of resting 4A5 is mostly low spin while the binding of laurate at a final concentration of 25 nM to resting enzyme converts the enzyme mostly to high-spin. These results were confirmed by substrate perturbation difference spectra with K, equal to 4.5 jiM. The measurement of lauric acid hydroxylation resulted in saturation kinetics and produced a Km of -9 nM and a turnover number of -41 min for the production of co-hydroxylaurate and a Km of -5 jiM and a turnover number of -11 mm ' for the production of ((o-l)-hydroxylaurate. No enzyme activity was detected with arachidonate.