Cytochrome P450 2C11:Escherichia coliExpression, Purification, Functional Characterization, and Mechanism-Based Inactivation of the Enzyme

The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16α-hydroxylase of the untreated rat liver. The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL). Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme. Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1%. For these reasons, we chose to heterologously express the enzyme inEscherichia coli.The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression inE. coli.The enzyme was isolated and purified fromE. colimembranes in relatively high yields (≈60%) and relatively high specific content (19 nmol/mg protein). The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL. Studies with14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both [22-14C]SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid. Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.