Structural Biology of Eicosanoid Biosynthesis

亚油酸和α-亚麻酸在哺乳动物生理学中起着极其重要的作用。 由于它们不能由哺乳动物合成，所有C-20、C-22和C-24多不饱和脂肪酸的生物合成取决于饮食中主要来自植物来源的必需脂肪酸的可用性。 一大组生物活性的含氧C-18、C-20和C-22脂肪酸（统称为类二十烷酸）衍生自n-3和n-6 PUFA。 内源性和外源性多不饱和脂肪酸的含氧产物对人体的许多生理过程具有有效和广泛的影响，包括细胞增殖、免疫反应、炎症、肾功能、生殖和心血管稳态。 这些化合物的区域和立体化学的细微变化也可以显著改变其生物学特性。 缺乏对导致立体化学上独特的含氧产物的化学以及这些产物如何转化为生物活性靶分子的清楚的分子理解。该更新申请的主要重点是通过X射线晶体学和诱变阐明（a）多不饱和脂肪酸的识别和结合的结构机制，（B）如何控制双氧合或单氧合的区域和立体化学，以及（c）区域和立体化学如何影响氧合的多不饱和脂肪酸的进一步代谢。 目前正在研究三类多不饱和脂肪酸及其含氧产物代谢中的酶：绵羊和人类前列腺素合成酶、大鼠前列环素合成酶以及植物中氧脂素代谢途径的3种相关酶。 这些酶利用血红素来激活底物，并且是完整的膜蛋白。 在前一个资助期，天然前列腺素合酶-1与结合花生四烯酸和其他脂肪酸的晶体结构被确定，重组前列腺素脱氢酶-1和-2的过表达系统被开发用于晶体学研究，并且超过50个前列腺素脱氢酶-1和-2的突变体被工程化用于结构分析。因此，可以研究双或单加氧反应的结构细节。 大鼠前列环素合酶和二乙烯基醚合酶的过表达也使得这两种膜结合的P450酶的晶体学研究易于处理。

The dietary or essential fatty acids linoleate and alpha-linolenate play extremely important roles in mammalian physiology. As they cannot be synthesized by mammals, the biosynthesis of all C-20, C-22 and C-24 polyunsaturated fatty acids depends on the availability of essential fatty acids in the diet, primarily from plant sources. A large group of bioactive, oxygenated C-18, C-20 and C-22 fatty acids, collectively known as eicosanoids, are derived from n-3 and n-6 PUFAs. The oxygenated products of polyunsaturated fatty acids, from endogenous and exogenous sources, have potent and wide-ranging effects on many physiological processes in humans including cell proliferation, immune responses, inflammation, renal function, reproduction, and cardiovascular homeostasis. Subtle changes in the regio- and stereochemistry of these compounds can also substantially alter their biological properties. A clear molecular understanding of the chemistry leading to stereochemically unique oxygenated products and how these products are converted into the bioactive target molecules is lacking. The primary focus of this renewal application is on elucidating, by X-ray crystallography and mutagenesis, (a) the structural mechanisms for the recognition and binding of polyunsaturated fatty acids, (b) how the regio- and stereochemistry of bis- or monooxygenation are controlled, and (c) how regio- and stereochemistry affect the further metabolism of the oxygenated polyunsaturated fatty acids. Three classes of enzymes in the metabolism of polyunsaturated fatty acids and their oxygenated products are being studied: sheep and human prostaglandin synthase, rat prostacyclin synthase, and 3 related enzymes of the oxylipin metabolic pathways in plants. These enzymes utilize heme to activate the substrates and are integral membrane proteins. In the previous grant period, the crystal structures of native prostaglandin synthase-1 with bound arachidonic acid and other fatty acids were determined, overexpression systems for recombinant prostaglandin synthases-1 and -2 were developed for crystallographic studies, and over 50 mutants of prostaglandin synthases-1 and -2 were engineered for structural analysis. Thus, the structural details of bis- or monooxygenation reactions can be studied. Overexpression of rat prostacyclin synthase and divinylether synthase also make crystallographic studies on these two membrane-bound P450 enzymes tractable.