Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca 2+-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 Å crystal structure of 8R-LOX and spectroscopic data suggested that Ca 2+ stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Á41-45:GSLOX) was engineered. Removal of the membraneinsertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 Å improvement in the resolution of the diffraction data.
A naturally occurring bi-functional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bi-functional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca 2+ -dependent membrane targeting of the sequential activities, despite the non-covalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca 2+ -dependent membrane binding relieves the non-covalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions. KeywordsEicosanoids; lipoxygenase; allene oxide synthase; bi-functional enzymes; C2-domains; Calciumdependent membrane binding; arachidonic acid; protein-protein interactions; Small angle X-ray scattering (SAXS); X-ray crystallographyThe ability of the cell to respond to its environment is dependent upon effective coordination of metabolic pathways. For those pathways that involve the biosynthesis of the arachidonic acid (AA) -derived lipid mediators such as the leukotrienes, prostaglandins, and thromboxanes, their coordination may be exceptionally challenging in terms of substrate acquisition and specificity. The hydrophobic substrates partition into the membrane phase, and active sites that recognize bulky hydrophobic compounds might be inherently promiscuous. Furthermore, the highly reactive fatty acid hydroperoxide intermediates promote cellular oxidative damage if their metabolism is not stringently regulated. Both compartmentalization of enzymes and organization of multi-enzyme complexes are thought to provide cellular mechanisms for "traffic control" in pathways for the synthesis of these potent signaling molecules (1-3). In order to understand how coordination of biosynthetic pathways is achieved in the context of *Author to whom correspondence should be addressed: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, Tel : (225) Fax: (225) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cell trafficking, and specifically how facilitated transfer of intermediates between active sites might be a means to regulate p...
Due to the various parameters that influence air solubility and microbubble production in dissolved air flotation (DAF), a multitude of values that cover a large range for these parameters are suggested for field systems. An unpacked saturator and an air quantification unit were designed to specify the effects of power, pressure, temperature, hydraulic retention time, and air flow on the DAF performance. It was determined that a pressure of 621 kPa, hydraulic retention time of 18.2 min, and air flow of 8.5 L/h would be the best controlled parameters for maximum efficiency in this unit. A temperature of 7 °C showed the greatest microbubble production, but temperature control would not be expected in actual application. The maximum microbubble flow from the designed system produced 30 mL of air (±1.5) per L of water under these conditions with immediate startup. The maximum theoretical dissolved air volume of 107 mL (±6) was achieved at a retention time of 2 h and a pressure of 621 kPa. To isolate and have better control over the various DAF operational parameters, the DAF unit was operated without the unsaturated flow stream. This mode of operation led to the formation of large bubbles at peak bubble production rates. In a real-world application, the large bubble formation will be avoided by mixing with raw unsaturated stream and by altering the location of dissolved air output flow.
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