No abstract

-The human cytochrome P450 4A11 is the major monooxygenase to oxidize the fatty acids and arachidonic acid. The production of 20-hydroxyeicosatetraenoic acid by P450 4A11 has been implicated in the regulation of vascular tone and blood pressure. Oxidation reaction by P450 4A11 requires its reduction partners, NADPH-P450 reductase (NPR). We report the functional expression in Escherichia coli of bicistronic constructs consisting of P450 4A11 encoded by the first cistron and the electron donor protein, NPR by the second. Typical P450 expression levels of wild type and several N-terminal modified mutants was observed in culture media and prepared membrane fractions. The expression of functional NPR in the constructed P450 4A11: NPR bicistronic system was clearly verified by reduction of nitroblue tetrazolium. Membrane preparation containing P450 4A11 and NPR efficiently oxidized lauric acid mainly to ω-hydroxylauric acid. Bicistronic coexpression of P450 4A11 and NPR in E. coli cells can be extended toward identification of novel drug metabolites or therapeutic agents involved in P450 4A11 dependent signal pathways.

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme. The recombinant CYP2W1 protein containing a 6 × (His) -tag at Nterminus has been expressed in Escherichia coli and purified. Expression level of CYP2W1 holoenzyme was around 500 nmol P450 holoenzyme per liter culture medium. The reduced CO difference spectrum of CYP2W1 showed a maximum absorption at 449 nm. CYP2W1 indicated the significant induction to bioactivate Trp-P-1, MeIQ, and IQ in E. coli DJ701 tester strain. However, the bioactivation of B[α]P, and NNK by CYP2W1 was relatively low. The model structure of CYP2W1 suggested the characteristic P450 folds with the lengths and orientations of the individual secondary elements. The F-G loop is situated on the distal side of heme to accommodate the flexibility of active site of CYP2W1. These studies can provide useful information for the finding of its biological roles and structure-function relationships of an orphan CYP2W1 enzyme.

An opportunistic fungal pathogen, Candida albicans contains the 10 putative cytochrome P450 (CYP) genes. They seem to play important roles for fungal survival and virulence. Here, we report the identification and characterization of CYP51, a putative lanosterol demethylase. The recombinant CYP51 protein containing a 6×(His)‐tag has been expressed in Escherichia coli and purified. The purified proteins showed the tight binding spectra with lanosterol. It exhibited a lanosterol demethylase activity in reconstitution with rat P450‐NADPH reductase to produce de methylated product in HPLC analysis. Azole compounds affinities of CYP51 enzyme was calibrated with econazole, itraconazole, ketoconazole and fluconazole. All four azoles showed Type II spectra with tight binding affinities to C. albicans CYP51. This study of functional characterization of CYP51 from C. albicans could provide the critical understanding of its role in the processing of pathogenesis. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009‐0088150).