| Lipoxygenases are a class of non-heme, non-sulfur iron dioxygenases in polyunsaturated fatty acid metabolism involved in the modulation of basic physiologic processes. The work described within this dissertation covers biochemical characterization of porcine leukocyte 12-lipoxygenase, and presents the crystal structure of its catalytic domain.;For the first time, the iron content of the full length protein was obtained as high as 0.94 atom per molecule. The loss of this iron atom from the protein was evident, as the pH declined from 5 to 4 without losing the native protein folding, by electrospray ionization mass spectroscopy measurements. The full length protein failed to yield crystals in the screening conditions using numerous site-directed mutagenesis experiments and multiple crystallization techniques.;As part of this study, the catalytic domain of porcine leukocyte 12-lipoxygenase was successfully expressed in E. coli cells. The crystal structure of porcine 12-lipoxygenase catalytic domain was determined to 1.89 Å as a complex with its specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) (PDB id: 3RDE). This represents the first structural description of the 12-lipoxygenase catalytic domain. The complex revealed a new one-open-end U-shaped channel for the natural substrate, arachidonic acid, which is remarkably different from the inhibitor (RS75091) binding pocket of rabbit 15-lipoxygenase.;Crystallization of the 12-lipoxygenase catalytic domain with arachidonic acid was also performed. Only fragments of electron density in the active site were present in the structure of the substrate-enzyme complex, illustrating that the substrate is not conformationally fixed in the crystal and may be converted into its products and derivatives. In addition, an unbound form of the catalytic domain was observed in the crystal structure of a recombinant manganese reconstituted lipoxygenase catalytic domain. Almost no structural differences were observed between the bound and unbound forms suggesting that lipoxygenase catalytic domain has little structural fluctuation or conformational alteration upon the binding of the inhibitor or substrate.;This research resulted in detailed biochemical insights for recombinant porcine leukocyte 12-lipoxygenase and its catalytic domain. The crystal structures of 12-lipoxygenase catalytic domain revealed key structural features of this enzyme, and also provided a basis for understanding enzymatic catalysis. |
