The cytochromes P450 and nitric oxide synthases are thiolate-ligated hemoproteins. The cytochromes P450 play key roles in metabolism of lipophilic xenobiotics and the biosynthesis of endogenous lipid factors, including 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is involved in regulation of vascular pressure. The nitric oxide synthases produce NO, a gaseous molecule with many functions as a neurotransmitter, vascoregulatory factor, and anti-infective agent. The two enzyme systems are linked by their mechanisms, the chemistry they catalyze, and the coordinate vascoregulatory actions of NO and 20-HETE. We propose to continue our structural, mechanistic, and functional analysis of both the cytochrome P450 and nitric oxide synthases. In the case of cytochrome P450, we plan to elucidate the structures of P450 enzyme active sites and their relationship to substrate specificity by mapping active site residues, preparing chimeras of mammalian and bacterial P450 enzymes, elucidating the structure and function of a thermophilic P450 (CYP119), and continuing an experimental and computational analysis of the determinants of substrate specificity. We will also carry out a detailed investigation of the CYP4A family of P450 enzymes by determining the basis for their chain length and the omega- hydroxylation regiospecificities, by developing CYP4A isoform-specific inhibitors that can be used in vivo to study the physiological roles of fatty acid omega-hydroxylation, by elucidating the differential substrate specificities of all the known rat and human CYP4A isoforms. In all parallel studies of the nitric oxide synthases we propose to investigate the structure and mechanism of the three nitric oxide synthase isoforms, with emphasis on the electron transfer pathway and the role of tetrahydrobiopterin, using chimeras, site specific mutagenesis, prosthetic group replacement, and sophisticated spectroscopic techniques. We will also characterize the dimer interfaces in the nitric oxide isoforms, clarify the contacts responsible for the differential binding of calmodulin to the three isoforms, and explore the possibility of using the dimer contact regions as a target for the development of isoform-specific nitric oxide synthase inhibitors.
| de Montellano, Paul R Ortiz (2018) 1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology. Med Chem (Los Angeles) 8: |
| Zhao, Yan; Wan, Debin; Yang, Jun et al. (2016) Catalytic Activities of Tumor-Specific Human Cytochrome P450 CYP2W1 Toward Endogenous Substrates. Drug Metab Dispos 44:771-80 |
| Conner, Kip P; Cruce, Alex A; Krzyaniak, Matthew D et al. (2015) Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1. Biochemistry 54:1198-207 |
| Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie et al. (2015) Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography. J Biol Chem 290:10000-17 |
| Varfaj, Fatbardha; Zulkifli, Siti N A; Park, Hyoung-Goo et al. (2014) Carbon-carbon bond cleavage in activation of the prodrug nabumetone. Drug Metab Dispos 42:828-38 |
| Frank, Daniel J; Madrona, Yarrow; Ortiz de Montellano, Paul R (2014) Cholesterol ester oxidation by mycobacterial cytochrome P450. J Biol Chem 289:30417-25 |
| Conner, Kip P; Schimpf, Alina M; Cruce, Alex A et al. (2014) Strength of axial water ligation in substrate-free cytochrome P450s is isoform dependent. Biochemistry 53:1428-34 |
| Nishida, Clinton R; Everett, Steven; Ortiz de Montellano, Paul R (2013) Specificity determinants of CYP1B1 estradiol hydroxylation. Mol Pharmacol 84:451-8 |
| Straub, Wesley E; Nishida, Clinton R; de Montellano, Paul R Ortiz (2013) Expression in Escherichia coli of a cytochrome P450 enzyme with a cobalt protoporphyrin IX prosthetic group. Methods Mol Biol 987:107-13 |
| Ortiz de Montellano, Paul R (2013) Cytochrome P450-activated prodrugs. Future Med Chem 5:213-28 |
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