Cytochrome P450 (P450) enzymes play key roles in cholesterol degradation in humans, initiating the conversion into bile acids and steroid hormones. Two microsomal and two mitochondrial enzymes, which have 25 percent amino acid sequence identity, bind cholesterol very specifically but introduce a hydroxyl group at different positions on either the steroid nucleus or the side chain. The long-term goals of this laboratory are: 1) to establish the structural basis by which cholesterol metabolizing P450s bind the very same substrate yet produce different products and 2) to identify in each P450 key amino acid residues that determine the remarkable regio and stereoselectivity of the hydroxylation reactions. The hypothesis to be tested is that two groups of residues determine substrate specificity in cholesterol hydroxylating P450s. The first group includes the residues located close to or on the surface of the molecule at the entrance of the substrate access channel. These residues mediate substrate recognition and at the same time perhaps play a role in anchoring the P450 to the membrane. The second group comprises the internal residues that hold cholesterol in place within the active site. The current proposal utilizes P450s 7A1 and 27A1 as models of microsomal and mitochondrial cholesterol metabolizing P450s, respectively.
The Specific Aims of the application are: 1) to identify in human P450s 27A1 and 7A1 internal active site amino acid residues important for cholesterol binding; 2) to identify the regions in human P450s 27A1 and 7A1 involved in association with the membrane; 3) to obtain high quality crystals of human P450s 27A1 and 7A1, thus allowing resolution of the atomic structures of these enzymes. Site-directed mutagenesis, heterologous expression in E. coli, assays of enzyme activity and substrate binding, and crystallographic methods will be used to achieve the goals of the project. Because P450s play such an important role in cholesterol homeostasis, the results will provide new biochemical insights into this essential biological process, will pave the way for future studies of other cholesterol metabolizng P45Os, and may contribute to the development of drugs that regulate plasma cholesterol levels. Furthermore, establishing a structural basis for high substrate specificity of two different cholesterol hydroxylating P45Os will lead to better understanding of general features of P450 structure/function.
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