Estrogens have long been implicated in the development and/or progression of certain forms of human cancer, including those of the endometrium and breast. In this context, the usefulness of estrogen antagonists as well as inhibitors of estrogen biosynthesis in the management of human breast cancer is well recognized. However, although estrogen receptor antagonists such as tamoxifin are efficacious in this context, there is a need to develop more effective and specific inhibitors of the biosynthesis of estrogens. The formation of estrogens from androgens is catalyzed by an enzyme complex known as aromatase which comprises a specific form of cytochrome P-450 (P-450AROM) and a flavoprotein, NADPH-cytochrome P-450 reductase. Our previous studies are indicative that a single form of P- 450AROM is present within human tissues and that its expression is encoded by a single gene. The objective of the present proposal is to understand the relationship of function to primary sequence and ultimately to three- dimensional structure of human P-450AROM. This is of particular significance for several reasons. In the first place, such knowledge will greatly facilitate the design of more effective and specific inhibitors of estrogen biosynthesis for use in the management of patients with breast cancer. Since there is only one form of P-450AROM in humans, any given aromatase inhibitor should be equally efficacious in inhibiting the enzyme regardless of the issue site of expression. Secondly, there is the need to understand the mechanism of the complex multi-step reaction sequence catalyzed by this enzyme. This goal will be addressed in the first instance by site-directed mutagenesis of a full-length cDNA insert encoding P-450AROM, and ultimately by the resolution of the three-dimensional structure of P-450AROM by means of X-ray crystallography. By means of site-directed mutagenesis, we will alter selected amino acids in domains of the protein which have been identified as being significant to P-450 catalyzed reactions in general. These selected amino acids are one which are unique to P-450AROM and which have been identified by computer-modeling of the substrate binding region as potentially important in the reaction mechanism of aromatase. We will also seek to express P-450AROM in unicellular organisms, namely E. coli and S. cerevisiae, as will as in baculovirus-infected insect cells, in order to produce large quantities of the enzyme as a starting point for purification and ultimately crystallization. Site-directed mutagenesis will be used as a means to attempt to express a soluble form of the enzyme in unicellular organisms such as E. coli and yeast in order to facilitate its purification and crystallization. This will be achieved by sequential alteration of condons for methionines at the amino-terminus, in order to delete the membrane- spanning region. Once optimum yields of P-450AROM have been obtained, then this material will be used to produce crystals of the enzyme which are suitable for analysis by X-ray spectrometry.
