CYTOCHROME-P450 2C11 - ESCHERICHIA-COLI EXPRESSION, PURIFICATION, FUNCTIONAL-CHARACTERIZATION, AND MECHANISM-BASED INACTIVATION OF THE ENZYME

The male-specific P450 enzyme CYP 2C11, whose expression is developmen tally and hormonally regulated, is the major steroid 16 alpha-hydroxyl ase of the untreated rat liver. The enzyme metabolizes a host of subst rates, including mechanism-based inactivators, such as rbethoxy-2,6-di methyl-4-ethyl-1,4-dihydro-pyridine (DDEP) and spironolactone (SPL), S tructural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully pu rified enzyme, Although several laboratories including our own have is olated and purified the enzyme from male rats, the yields are typicall y low and of the order of 1%. For these reasons, we chose to heterolog ously express the enzyme in Escherichia coli. The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vecto r pcW after appropriate modifications for optimal expression in E. col i. The enzyme was isolated and purified from E. coli membranes in rela tively high yields (approximate to 60%) and relatively high specific c ontent (19 nmol/mg protein). The purified recombinant enzyme had spect ral and functional characteristics comparable to those reported for th e native rat liver enzyme, including mechanism-based inactivation by D DEP and SPL. Studies with C-14-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the othe r hand, studies with radiolabeled SPL-SH (the proximal inactivating de acetylated metabolite of SPL) revealed that although both [22-C-14]SPL -SH and SPL-(SH)-S-35 inactivated the enzyme, only SPL-(SH)-S-35 was f ound to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thi ol 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 c onsiderably differ from the corresponding modes of P450 3A inactivatio n by these agents, wherein heme modification of the protein predominat es. (C) 1997 Academic Press.