title: A comparative approach to structure-function studies of mammalian aromatases.
abstract: To date, structure--function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B' and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.
authors.affiliation: Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. ajconley@ucdavis.edu
authors.forename: A
authors.initials: A
authors.lastname: Conley
authors.forename: S
authors.initials: S
authors.lastname: Mapes
authors.forename: C J
authors.initials: CJ
authors.lastname: Corbin
authors.forename: D
authors.initials: D
authors.lastname: Greger
authors.forename: K
authors.initials: K
authors.lastname: Walters
authors.forename: J
authors.initials: J
authors.lastname: Trant
authors.forename: S
authors.initials: S
authors.lastname: Graham
mesh_terms: D000818:Animals; D001141:Aromatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047072:Aromatase Inhibitors; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005045:Etomidate / Q000494:pharmacology; D017316:Fadrozole / Q000494:pharmacology; D005260:Female; D006066:Gonads / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013552:Swine; D014162:Transfection
chemical_list: D047072:Aromatase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D001141:Aromatase; D017316:Fadrozole; D005045:Etomidate