This proposal is based upon the applicants' recent cloning of the rat 1-hydroxylase (cytochrome P450c1) enzyme and the human P450c1 gene. Using FISH analysis and human P450c1 DNA, they have determined the gene locus for P450c1 to be 12q13.1-13.3, the same site that was previously identified for pseudovitamin D-deficient rickets (PDDR). This application describes studies that are directed toward understanding the structure and function of P450c1, a regulatory enzyme that directs the bioactivation of vitamin D to the pleiotropic hormone 1,25-dihydroxy D3 [1,25(OH)2D3 or calcitriol], which is active in calcium homeostasis, cellular growth and differentiation, and the immune response. However, until recent advances, only a paucity of information existed on the molecular attributes of the 1-hydroxylase enzyme due to the lack of pure protein and enzyme coding information. Yet, a molecular understanding of the enzyme's binding and catalytic properties is fundamental to the biostructural design of 1-hydroxylase inhibitors for application in hyperparathyroidism and 1,25(OH)2D3-mediated hypercalcemic states. In addition, it is now possible to use the P450c1-gene information to investigate the molecular basis of PDDR, and in the design of gene therapeutic agents for the treatment of genetic disorders and cellular proliferation diseases (e.g., cancer). Therefore, the current investigation is designed to gain an understanding of the structural and functional attributes of cytochrome P450c1 in health and disease. It is hypothesized that a structural defect in P450c1 is the basis for PDDR, and that delineation of the genetic mutations in this disease will serve as a foundation for understanding the molecular function of cytochrome P450c1. Studies in the Specific Aims are designed to: 1) express and purify recombinant cytochrome P450c1, prepare antibodies and determine substrate-binding, spectral, kinetic and topological properties of the enzyme, and 2) to sequence analyze, and characterize the activity and substrate-binding properties of expressed natural mutants of the human and porcine.
