Analytical approaches to fatty acid oxygenases and their lipid mediator products

Analytical approaches to fatty acid oxygenases and their lipid mediator products Project Summary/Abstract This program focuses on technically challenging questions related to lipoxygenases, their associated enzymes and proteins, and an unprecedented higher-resolution analysis of mechanisms of biosynthesis and fate of the oxidation products. These pathways are conserved widely in evolution and ubiquitous in higher animals. A major thrust is uncovering the mechanistic basis of how a 12R-lipoxygenase (12R-LOX) metabolic pathway functions to secure formation of the water permeability barrier in the epidermis. Pathway genetic deficiencies have devastating consequences, being neonatal lethal in mice and in humans causing congenital ichthyosis (scaly skin), an extremely socially challenging condition. We identified 12R-LOX, epidermal-LOX-3, and the dehydrogenase SDR9C7, as working in series to oxidize linoleic acid (18:2w6) that is esterified in a skin-specific acylceramide. Such oxidations are essential for covalent binding of ceramides to barrier protein, forming sub- structures visible by EM: the corneocyte lipid envelopes. We will apply a singular combination of technical resources (recombinant enzymes and barrier proteins, novel LOX products, unique collection of oxidize acylceramides, access to mouse knockout epidermis, quantitative LC-MS assays, proteomics) to uncover how 12R-LOX oxidations lead to sealing the permeability barrier. Our hypothesis rides on the nature of the linoleate- ester oxidation, which forms an epoxy-enone-linoleate, reactive with nucleophiles and capable of adducting to protein. Understanding these mechanisms will have long-term impact on treatment of skin barrier-related diseases as well as optimizing artificial skin production for treating burns and severe diabetic ulcers. In a second arm of the program, we address technical questions in the field of lipoxygenase product biosynthesis. First, defining the biochemical mechanisms of forming the oxygenated derivatives of “fish oil” fatty acid EPA and DHA, products currently cited as pro-resolving in inflammation, the biosynthesis remaining controversial over the past 20 years. Second, the mechanisms in cyclization of fatty acid allene oxides (highly unstable epoxides) to cyclopentenones. The latter is relevant to heme-protein chemistry and to synthetic chemistry of cyclopentenones and other 5-membered carbocycles. Summing up the vision and suitability for the freedom afforded by MIRA funding: we address mechanistically important and challenging projects, apply unparalleled technical abilities including analysis of extremely unstable pathway intermediates, and leadership in exemplifying the highest standards in the fields of lipoxygenases and related lipid oxygenases.