STRATEGIES TO ENHANCE THE COEXPRESSION OF CYTOCHROME P450 2E1 AND REDUCTASE IN BACTERIA*

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Notes

*This paper was refereed by F. Peter Guengerich Ph.D., Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232.

*Flavodoxin with flavodoxin reductase has been shown to catalyze electron transfer from NADPH to some forms of mammalian P450 in reconstituted systems, and probably in vivo [17]; however, activity is low, and unlike P450 reductase, this redox pathway is not membrane bound and thus does not cosegregate with P450 in the isolated membrane preparations often used for in vitro assays.

*This may also reflect differences in culture conditions, as well as methodological differences in the preparation of bacterial membranes and in the calculation of P450 yields. Although we have previously suggested [16] that use of a French press to prepare bacterial membranes may be less efficient than sonication, in the present studies we compared yields using both techniques, following the sonication protocol of Gillam et al. [2], and obtained similar yields by both methods. Both laboratories use a rich culture medium and similar culture conditions, although we do not add supplements other than δ-aminolevulinic acid and 4-methylpyrazole to our media.