DESCRIPTION (provided by applicant): The large cytochrome P450 (P450, CYP) superfamily is distributed throughout all biological kingdoms. The two general classes of P450 reactions are xenobiotic metabolism (e.g., drugs, carcinogens) and endogenous substrate biosynthesis (e.g., hormones, eicosanoids). High-resolution structures of members of ~50 subfamilies among the >17,000 P450 superfamily members are known (all showing a common structural fold), but the precise roles of most individual structural elements are not clearly established. We propose to address this topic by investigating two P450s involved in steroid hormone biosynthesis: mammalian P450 21A2, which is known to have >100 amino acid mutations that influence its steroid 21-hydroxylation activity, and human P450 17A1 and zebrafish P450s 17A1 and 17A2, the latter two being distinct enzymes with dramatic variations in their steroid 17α-hydroxylation/17,20-lyase activities. By analyzing these enzymes in detail (structure and kinetics), we will be able to establish a much more precise view of P450 structure/function than is currently available. We anticipate that these studies will clarify in detail how these two important P450s can function in steroid hormone biosynthesis, and the results can then be applied to understanding the structure/function of other P450s. Mutations in both P450 21A2 and 17A1 are the major causes of a genetic group of diseases known as congenital adrenal hyperplasia, and P450 17A1 is an important drug target for treatment of prostate cancer. The results from these studies will allow a better understanding of both diseases. Taking advantage of determining how amino acid alterations modify functional properties of these enzymes, we will identify the roles that specific structural regions play. The methodology will involve heterologous protein expression/purification/mutagenesis, X-ray crystallography, detailed kinetic analysis, and other biochemical studies (e.g., binding of substrates and other proteins). It is expected that these innovative studies will produce novel insights into and unexpected new understanding of P450 structure/function.

PUBLIC HEALTH RELEVANCE: Of the more than 17,000 known cytochrome P450 (CYP) genes, two steroid hydroxylases show particularly interesting properties that can lead to more detailed understanding of P450 structure and function. P450 21A2 (21-hydroxylase), required for glucocorticoid/mineralocorticoid biosynthesis, has the largest number of naturally occurring variations (>100) that alter activity and P450 17A1 (17α-hydroxylase/17,20-lyase) catalyzes two different activities, depending on its cellular location. This project will take advantage of these unique variations in the two P450s to explain their structure/function relationships in detail and also establish a better understanding of the general basis of P450 function.