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. 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 int he 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. Lipoxygenase are being investigated as possible therapeutic agents in all of these areas. In this project we propose to investigate the mechanism of the lipoxygenase reaction and the nature of enzyme/substrate interactions by studying the atomic structure of soybean lipoxygenase-1 and of its complexes. 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. In the first period of this project we have obtained crystals of soybean lipoxygenase-1, and determined and refined the three-dimensional structure of the enzyme. We have, in addition, generated, by homology modeling, models of human 5-,12- and 15-lipoxygenases. In the present application we propose to: (1) determine the structure of the Fe+3 form of soybean lipoxygenase-1, (2) analyze the mode of binding of oxygen to the enzyme using nitric oxide and azide, (3) investigate the mechanism of lipoxygenases by determining the structures of complexes of Ll with selected substrates, products and analogs, (4) analyze the mode of action of compounds design to inhibit mammalian lipoxygenases, (5) map the location of substrates and products in the internal cavities of Ll using Xenon as an x-ray contrast agent.
