The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

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This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.

"Personalized medicine" is a new concept in health care, one aspect of which defines the specificity and dosage of drugs according to effectiveness and safety for each patient. Dosage strongly depends from the rate of metabolism which is primarily regulated by the activity of cytochrome P450. In addition to the need for a genetic characterization of the patients, there is also the necessity to determine the drug-clearance properties of the polymorphic P450 enzyme. To address this issue, human P450 2D6 and 2C9 were engineered and covalently linked to an electrode surface allowing fast, accurate, and reliable measurements of the kinetic parameters of these phase-1 drug metabolizing polymorphic enzymes. In particular, the catalytic activity of P450 2C9 on the electrode surface was found to be improved when expressed from a gene-fusion with flavodoxin from Desulfovibrio vulgaris (2C9/FLD). The results are validated using marker drugs for these enzymes, bufuralol for 2D6, and warfarin for 2C9/FLD. The platform is able to measure the same small differences in K(M), and it allows a fast and reproducible mean to generated the product identified by HPLC from which the k(cat) is calculated.