Structural Biology of Eicosanoid Biosynthesis

膳食或必需脂肪酸亚油酸和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必需脂肪酸的可用性，主要来自植物来源。一大类生物活性的、含氧的C-18、C-20和C-22脂肪酸，统称为二十碳酸类化合物，来自n-3和n-6多不饱和脂肪酸。多不饱和脂肪酸的氧化产物来自内源性和外源性，对人类的许多生理过程具有强大而广泛的影响，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极大地改变它们的生物学特性。对导致立体化学独特的氧化产物的化学以及这些产物如何转化为生物活性目标分子的化学缺乏明确的分子理解。这一更新应用的主要焦点是通过X射线结晶学和诱变，阐明(A)多不饱和脂肪酸识别和结合的结构机制，(B)双加氧或单加氧的区域和立体化学是如何控制的，以及(C)区域和立体化学如何影响含氧多不饱和脂肪酸的进一步代谢。目前正在研究多不饱和脂肪酸及其氧化产物代谢中的三类酶：绵羊和人前列腺素合成酶、大鼠前列腺素合成酶和植物氧化脂质代谢途径的3种相关酶。这些酶利用血红素激活底物，是完整的膜蛋白。在之前的资助期间，已经确定了天然前列腺素合成酶-1与花生四烯酸和其他脂肪酸结合的晶体结构，开发了用于结晶学研究的重组前列腺素合成酶-1和-2的高表达系统，并设计了50多个前列腺素合成酶-1和-2的突变体用于结构分析。因此，可以研究双氧反应或单氧反应的结构细节。大鼠前列环素合成酶和二乙烯基醚合成酶的过表达也使这两种膜结合的P450酶的结晶学研究变得容易。

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.