This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Arachidonic acid serves as a precursor to numerous biologically active compounds that include the prostaglandins, leukotrienes and thromboxanes, collectively known as eicosanoids. Eicosanoids are known as local hormones, as they exert their effects on cells close to their sites of synthesis and are rapidly degraded. The intermediates are both labile and exhibit limited solubility; and one way to optimize acquisition of a hydrophobic or labile substrate is to associate enzymes of the metabolic pathways either covalently such as in mammalian fatty acid synthase, or non-covalently. A naturally occurring 8R-lipoxygenase (8R-LOX) and allene oxide synthase (AOS) fusion protein from Plexaura homomalla catalyses successive steps in a biosynthetic pathway. The intermediate in the reaction, the 8R-hydroperoxy derivative of arachidonic acid, is both hydrophobic and labile. The structure of the AOS-LOX fusion protein is essential in understanding how an assembly of catalytic activities impacts the transfer of the reaction intermediate, and thus the efficiency of overall biosynthesis. We have determined the crystal structures of the independently expressed domains and have shown that they associate into a heterodimeric macromolecular complex, which is not amenable to crystalization. We thus wish to determine the structure of the AOS-LOX complex by small angle x-ray scattering. We have made preliminary solution x-ray scattering measurements to evaluate the feasibility of this approach and have obtained data that indicate that the proteins are stable in the x-ray beam. The data also implies difference in crystal and solution structures for AOS while LOS solution structure seems consistent with its crystal structure as well as one of the previous x-ray scattering studies.
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