Research Interests

Crystallographic characterization and functional analysis of integral membrane enzymes involved in lipid metabolism; fatty acid desaturation; enzymatic mechanisms of fatty acid oxygenation; development of tools for high-throughput structural characterization of membrane proteins.

The Structural Biology of Oxylipin Biosynthesis

Public perception of healthy food and lifestyle, as well as the identification of specific biological roles in certain clinical conditions, has brought a renewed attention to polyunsaturated fatty acids (PUFAs) with respect to health and disease.  PUFAs are lipid molecules that differ in hydrocarbon length and degree of saturation.  These essential fatty acids cannot be synthesized de novo by most eukaryotic organisms and therefore must be obtained from dietary sources.  Once ingested, PUFAs are acted upon by desaturase and elongase enzymes, which form a larger family of precursors that play a key role in maintaining the fluidity and structural integrity of all cell membranes.  PUFAs play an equally significant role as signaling molecules in numerous biologically vital pathways.  However, in order to exert their full range of biological effects, PUFAs are generally metabolized into more potent substances called oxylipins. The term “oxylipin” is used collectively to refer to oxygenated, 18-22 carbon PUFA-based compounds that are biosynthesized through the stereospecific addition of one, two, or four atoms of oxygen at different carbon positions, followed by subsequent conversion of the developing peroxide or hydroperoxide intermediate into a stable, biologically active compound.  Arachidonic acid (AA; 20:4 w-6) is the most vital PUFA precursor of the eicosanoid class of oxylipins in animals.  The “arachidonate cascade”, which is comprised of the cyclooxygenase, lipoxygenase, and epoxygenase pathways, is responsible for the production of prostaglandins, leukotrienes, lipoxins, hydroxy-, and epoxy- eicosatetraenoic acids.  These potent lipid signaling molecules are synthesized and released rapidly in response to extracellular stimuli, where they play an important role in a number of physiological processes.  Moreover, they play a major role in a vast array of inflammatory disorders  such as asthma, rheumatoid arthritis, atherosclerosis, and can be linked to other diseases that are associated with inflammation including cardiovascular disease, Alzheimer's disease, and cancer.

The major focus of my research is to use biochemical analyses, biophysical tools, and x-ray crystallographic methods to understand how different classes of integral membrane enzymes carry out stereoselective oxygenations to generate unique oxylipins from a defined set of PUFA substrates.  To gain insight on the chemical determinants that drive stereoselective oxygenation of PUFAs at the molecular level, we are studying different integral membrane enzymes involved in oxylipin production in plants, animals, and invertebrates.

Understanding the Stereoselective Oxygenations of AA by Aspirin-Acetylated COX-2

Our goal is to understand the molecular mechanism by which Cyclooxygenase-2 (COX-2) stereospecifically oxygenates AA to form "novel" (15R)-prostaglandins (15R-PGs).  COX-1 and COX-2 catalyze the committed step in PG biosynthesis and are the pharmacological targets of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), which provide relief from the inflammation, swelling, and pain associated with arthritis.  In contrast to all other NSAIDs, aspirin covalently modifies COX-1 and COX-2 via the acetylation of a critical serine residue within the cyclooxygenase active site, resulting in complete inhibition of PG biosynthesis.  However, aspirin acetylation of COX-2 results in a shift in the reaction specificity of the enzyme, converting it from a cyclooxygenase to a lipoxygenase, resulting in the formation of (15R)-hydroxyeicosatetraenoic acid (15R-HETE).  15R-HETE is converted by downstream enzymes into (15R)-epi lipoxin, an aspirin-triggered lipid mediator with potent anti-inflammatory actions.  We are using a combination of site-directed mutagenesis, functional analyses, and x-ray crystallographic methods to test the hypothesis that the inversion of stereochemistry at carbon-15 observed in products formed by aspirin acetylated COX-2 is a result of AA binding in an "unconventional" conformation within the cyclooxygenase active site prior to the initiation of catalysis.  These studies will provide for a complete mechanistic understanding of how 15R-PGs (and ultimately aspirin-triggered lipoxins) are generated, lend valuable insight into how COX enzymes stereospecifically control oxygenation during catalysis, and lead to further exploration and development of new or combined therapeutic approaches for the treatment of arthritis, with fewer unwanted side effects.

Characterizing the Structural Determinants Involved in the Stereoselective Oxygenation of 18-Carbon PUFAs by Pathogen Inducible Oxygenase (PIOX), a Plant Fatty Acid Alpha Dioxygenase

Pathogen-Induced Oxygenase (PIOX) is a heme-containing, membrane-associated protein found in mono- and dicotyledon plats that  utilizes molecular oxygen to convert the PUFAs linoleic acid and  alpha linolenic acid into their corresponding 2R-hydroperoxides.  PIOX, which is a key component of the host-defense response in plants, is a member of a larger family of fatty acid alpha dioxygenases that includes mammalian COX-1 and COX-2.  Many residues crucial for activity in mammalian COX are conserved in PIOX.  These residues include the distal and proximal histidine residues involved in heme binding, and a catalytic tyrosine, which is responsible for the initiation of catalysis.  While there are global differences between PIOX and COX enzymes with respect to their potential overall structural architecture, substrate specificities, and signaling pathways, the mechanism by which these enzymes stereoselectively oxygenate PUFAs are strikingly similar.  Our goal is to elucidate the structure of PIOX and determine the mechanism controlling catalysis.  This information will enhance the existing knowledge of how PUFAs are stereoselectively oxygenated and provide useful insights into other systems, which discriminate between the saturation and chain length of a PUFA substrate.