Soybean lipoxygenase-1 is highly homologous to mammalian lipoxygenases - a family of non-heme, iron-containing dioxygenases that catalyze the hydroperoxidation of arachidonic acid. In animals, this reaction is the first step in the biosynthetic cascade that generates leukotrienes and lipoxins, two classes of potent physiological effectors. The leukotrienes participate in host defense reactions, as well as in pathophysiological conditions such as immediate hypersensitivity, inflammation and asthma. Lipoxins have physiological effects that range from lung contraction and microvascular dilation to the inhibition of natural killer-cell cytotoxicity. Thus, lipoxygenases are key enzymes in the generation of compounds that regulate important cellular responses in inflammation and immunity. In addition, human 15-lipoxygenase has been found in association with oxidized low-density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Inhibitors of these enzymes are being investigated as possible therapeutic agents in all of these areas. In this project we propose to investigate the atomic structure of soybean lipoxygenase-1 and of its complexes, in order to evaluate the coordination of the iron with its ligands and to gain insight into the nature of enzyme/substrate interactions and the mechanism of the reaction. Soybean lipoxygenase-1 is relatively easy to purify, sufficiently stable and can be obtained in large quantities. Given the sequence similarities among the different enzymes, it is expected that many of the conclusions obtained for the soybean enzyme can be extended to other mammalian systems. We have obtained crystals of soybean lipoxygenase-1 and have determined and refined the three-dimensional structure of the enzyme. We propose to: 1) refine the model using data to 2.5 Angstrom resolution, 2) determine the structure of the complex of lipoygenase-1 with nitric oxide, 3) determine the structure of the enzymes in complexes with substrate analogs, inhibitors and products, 4) determine the structure of the enzyme in complexes with inhibitors of the mammalian enzymes, 5) model the structures of mammalian 5-, 12-, and 15-lipoxygenases and attempt to design inhibitors of mammalian lipoxygenases.
