Author/Authors :
Richardson، نويسنده , , Toby H. and Jung، نويسنده , , Frank and Griffin، نويسنده , , Keith J. and Wester، نويسنده , , Mike and Raucy، نويسنده , , Judy L. and Kemper، نويسنده , , Byron and Bornheim، نويسنده , , Lester M. and Hassett، نويسنده , , Christopher and Omiecinski، نويسنده , , Curtis J. and Johnson، نويسنده , , Eric F.، نويسنده ,
Abstract :
Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs inEscherichia colias chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3′-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of δ-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed inE. coliwere concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16α-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17α- and 16α-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbu tamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s inE. coli.The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.
