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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045455-04
Application #
6375145
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Sharrock, William J
Project Start
1998-07-22
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
4
Fiscal Year
2001
Total Cost
$248,158
Indirect Cost
Name
University of New Mexico
Department
Biochemistry
Type
Schools of Medicine
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Annalora, Andrew J; Bobrovnikov-Marjon, Ekaterina; Serda, Rita et al. (2007) Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function. Arch Biochem Biophys 460:262-73
Astecker, Norbert; Bobrovnikova, Ekaterina A; Omdahl, John L et al. (2004) C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1. Arch Biochem Biophys 431:261-70
Annalora, Andrew; Bobrovnikova-Marjon, Ekaterina; Serda, Rita et al. (2004) Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity. Arch Biochem Biophys 425:133-46
Chavez, L S; Serda, R; Choe, S et al. (2003) Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig. J Nutr Biochem 14:378-85
Omdahl, J L; Bobrovnikova, E A; Choe, S et al. (2001) Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway. Steroids 66:381-9