The broader goal of this proposal is to understand how different integral membrane enzymes utilize stereoselective oxygenations to generate unique oxylipins from a defined set of polyunsaturated fatty acid (PUFA) substrates. Oxylipins are bioactive lipid mediators that are biosynthesized from 18-22 carbon PUFAs through the addition of molecular oxygen via the catalytic activity of cytochrome P450s, lipoxygenases, and cyclooxygenases (COX-1 and COX-2). One of the most biologically important groups of oxylipins is the eicosanoid class, which include prostaglandins (PGs) and leukotrienes derived from arachidonic acid. These products are responsible for the modulation of basic physiologic processes and act as potent lipid mediators of the inflammatory process and other immune responses. COX-1 and COX-2 are the pharmacological target of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), including the COX-2 specific inhibitors Vioxx, Bextra, and Celebrex.
SPECIFIC AIMS : Using mutagenesis, functional analyses, and x-ray crystallographic methods, we will (1) elucidate the structure of pathogen-inducible oxygenase and characterize at the molecular level the mechanism and structural determinants involved in the stereoselective oxygenation of 18 carbon PUFAs into 2R-oxylipin products;and (2) elucidate the structures of unique 15R-PG producing COX:PUFA complexes in order to understand at the molecular level how the conformation of the PUFA in the active site influences stereospecific oxygenation in the generation of these novel products.
The role that PUFAs play in health and disease is generating renewed interest, with a more focused public perception of healthy food and lifestyle and the significant impact that these compounds have in certain clinical conditions. The ability of proteins, such as COX-2, to dramatically shift their product profiles upon treatment with pharmacological inhibitors has led to further investigations into how these enzymes function. Our studies will provide for a complete mechanistic understanding of how novel lipids are derived from PUFAs upon aspirin treatment, and lend valuable insight into development of new or combined therapeutic approaches for the management of arthritis and vascular inflammation, with fewer unwanted side effects.
|Dong, Liang; Yuan, Chong; Orlando, Benjamin J et al. (2016) Fatty Acid Binding to the Allosteric Subunit of Cyclooxygenase-2 Relieves a Tonic Inhibition of the Catalytic Subunit. J Biol Chem 291:25641-25655|
|Orlando, Benjamin J; Malkowski, Michael G (2016) Crystal structure of rofecoxib bound to human cyclooxygenase-2. Acta Crystallogr F Struct Biol Commun 72:772-776|
|Lucido, Michael J; Orlando, Benjamin J; Vecchio, Alex J et al. (2016) Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 55:1226-38|
|Orlando, Benjamin J; Lucido, Michael J; Malkowski, Michael G (2015) The structure of ibuprofen bound to cyclooxygenase-2. J Struct Biol 189:62-6|
|Orlando, Benjamin J; Borbat, Peter P; Georgieva, Elka R et al. (2015) Pulsed Dipolar Spectroscopy Reveals That Tyrosyl Radicals Are Generated in Both Monomers of the Cyclooxygenase-2 Dimer. Biochemistry 54:7309-12|
|Orlando, Benjamin J; McDougle, Daniel R; Lucido, Michael J et al. (2014) Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs. Arch Biochem Biophys 546:33-40|
|Goulah, Christopher C; Zhu, Guangyu; Koszelak-Rosenblum, Mary et al. (2013) The crystal structure of ?-Dioxygenase provides insight into diversity in the cyclooxygenase-peroxidase superfamily. Biochemistry 52:1364-72|
|Zhu, Guangyu; Koszelak-Rosenblum, Mary; Malkowski, Michael G (2013) Crystal structures of ?-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide. Protein Sci 22:1432-8|
|Vecchio, Alex J; Orlando, Benjamin J; Nandagiri, Ritwik et al. (2012) Investigating substrate promiscuity in cyclooxygenase-2: the role of Arg-120 and residues lining the hydrophobic groove. J Biol Chem 287:24619-30|
|Vecchio, Alex J; Malkowski, Michael G (2011) The structural basis of endocannabinoid oxygenation by cyclooxygenase-2. J Biol Chem 286:20736-45|
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