Polyunsaturated fatty acid metabolism has important consequences for human health. Arachidonic acid, the primary polyunsaturated fatty acid in humans, is converted in the body into a host of bioactive compounds referred to collectively as eicosanoids. There is compelling evidence that these metabolic pathways have a major impact on the leading causes of death in humans, cardiovascular disease and cancer. Two enzymes, cyclooxygenase and lipoxygenase inaugurate polyunsaturated fatty acid metabolism. The focus of the current proposal is the lipoxygenase-initiated pathway. There are six distinct lipoxygenase genes in humans leading to a family of enzyme isoforms with unique properties and cellular distributions. The broad, long term objective of this research is to gain a sufficiently detailed understanding of the structure and mechanism of action of this class of enzymes to allow for the design and synthesis of lipoxygensase isoform-specific inhibitors. The lipoxygenases are mediators of early inflammatory events in atherosclerosis, and a genetic variation in a specific lipoxygenase gene promoter represents a risk factor similar in magnitude to diabetes for the disease in humans. In cancer, some lipoxygenases have procarcinogenic effects while others exhibit anticarcinogenic effects. These findings highlight the importance of gaining a detailed understanding of structure and mechanism for this class of enzymes. It will be necessary to compare and contrast the structures and properties of these closely related lipoxygenase isoforms in order to contemplate the design of specific inhibitors with therapeutic potential. The first two specific aims of this proposal are to produce, isolate, and crystallize two 12-lipoxygenases. This is an essential objective for the determination of the structures by X-ray crystallography. We will use both robotic and manual procedures. The chemistry department at the University of Toledo is the home of the Ohio Crystallography Consortium, and as such we are exceptionally well equipped to conduct these experiments. Lipoxygenases are unusual among enzymes; they are activated by the product of the catalyzed reaction. With two states, active and inactive, there is an opportunity to control catalysis by manipulating the balance between the two. The third and fourth specific aims of the proposal are to identify the product of a new reaction we recently observed between the enzyme and product using electrospray ionization mass spectrometry and to explore the scope of this reaction. For this, we will use extraction, derivatization and GC-MS, as well as 1H-NMR spectroscopy. In a fifth specific aim, we will investigate the scope of the activation reaction with compounds similar to the product, but with different molecular structures.
This aim will require the synthesis and characterization of several compounds using organic chemistry, e.g. the Grignard reaction, and the determination of their effect on the reaction catalyzed by lipoxygenase using a kinetic assay. The primary objective of this proposal is to provide research opportunities for undergraduates in projects that employ concepts and laboratory experiences from the chemistry and biology curricula. ? ? ? ? ?
| Xu, Shu; Mueser, Timothy C; Marnett, Lawrence J et al. (2012) Crystal structure of 12-lipoxygenase catalytic-domain-inhibitor complex identifies a substrate-binding channel for catalysis. Structure 20:1490-7 |
| Arif, Waqar; Xu, Shu; Isailovic, Dragan et al. (2011) Complexes of the outer mitochondrial membrane protein mitoNEET with resveratrol-3-sulfate. Biochemistry 50:5806-11 |
| Rapp, Johanna; Xu, Shu; Sharp, Allan M et al. (2009) EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase. Arch Biochem Biophys 490:50-6 |
