FATTY ACID SYNTHASE: STRUCTURE, DYNAMICS AND FUNCTION

产品描述（申请人提供）：长期研究目标是确定人脂肪酸合成酶（FAS）的三维结构、动力学性质和功能，FAS是一种催化脂肪酸合成的酶，其高活性与包括乳腺癌、前列腺癌、结肠癌和卵巢癌在内的多种人类恶性肿瘤密切相关。这一目标需要详细的机械理解的同源二聚体动物FAS的约0.54万道尔顿进行七个连续的反应和引导的底物的生物合成的长链脂肪酸。目前的信息学研究表明，FAS不仅具有多个催化结构域，而且具有高的运动性和多种构象。这些特点，加上其大尺寸，使FAS结构功能研究的独特吸引力和挑战性的系统。为了迎接这一挑战，已经组建了一个具有电子冷冻显微镜（cryo-EM），X射线晶体学，计算生物物理学和酶学专业知识的研究小组。该小组将采取一种系统的方法，通过结合从各种技术中获得的数据，以尽可能高的分辨率阐明FAS结构。由于存在多构象的FAS，一种新的混合实验和计算的细化程序将采用迭代合并结构和计算信息学在不同的分辨率，以产生三维结构的酶在逐步更详细。此外，在配体存在下，单个或串联功能结构域的晶体结构的精确原子细节将在理解催化机制和未来合理设计抗癌疗法方面具有非常宝贵的价值。具体目标是：1）通过冷冻-EM最终确定FAS至10 A的三维结构，并通过新的计算细化程序增强。2)通过X射线晶体学确定FAS的催化/功能结构域的原子结构，单独或串联。3)通过抗体Fab片段和/或金标记，并将结构域的晶体结构与完整酶的冷冻电镜图拟合，绘制出结构域的位置。4)整合所有结构、计算和生物化学信息学，以推导FAS作用模式的分子基础，从而导致长链脂肪酸的多步催化合成。

DESCRIPTION (provided by applicant): The long-term research goal is to determine the three-dimensional structure, dynamic properties and function of human fatty acid synthase (FAS), an enzyme which catalyzes the synthesis of fatty acids, and whose high activity is closely related to many human malignant tumors including breast, prostate, colon and ovarian carcinoma. This goal requires a detailed mechanistic understanding of how the homodimeric animal FAS of approximately 0.54 million Dalton carries out seven consecutive reactions and channeling of substrates for the biosynthesis of long chain fatty acids. Current informatics indicate that FAS, not only consists of multi-catalytic domains, but also exhibits high mobility with multiple conformations. These features, combined with its large size, make FAS a uniquely attractive and challenging system for structure-function studies. To meet the challenge, a team of investigators with expertise in electron cryo-microscopy (cryo-EM), x-ray crystallography, computational biophysics and enzymology has been assembled. The team will undertake a systematic approach to elucidate the FAS structure at the highest possible resolution, by combining data obtained from the various techniques. Because of the existence of multi-conformers of FAS, a novel hybrid experimental and computational refinement procedure will be employed iteratively to merge structural and computational informatics at various resolutions, in order to generate three-dimensional structures of the enzyme at progressively greater detail. Furthermore, the precise atomic details of the crystal structures of individual or tandem functional domains in the presence of ligands will be invaluable in understanding catalytic mechanisms and in the future rational design of anti-cancer therapeutics. The specific aims are: 1) To determine, ultimately, the three-dimensional structure of FAS to 10 A by cryo-EM, augmented by a novel computational refinement procedure. 2) To determine the atomic structures of catalytic/functional domains, individually or in tandem, of FAS by x-ray crystallography. 3) To map out the locations of the domains by antibodyFab fragments and/or gold labeling and fitting crystal structures of domains to cryo-EM maps of the intact enzyme. 4) To integrate all the structural, computational and biochemical informatics, in order to derive a molecular basis for the mode of action of FAS leading to the multi-step catalytic synthesis of long chain fatty acid.