FATTY ACID SYNTHASE: STRUCTURE, DYNAMICS AND FUNCTION

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，其高活性与乳腺癌、前列腺癌、结肠癌、卵巢癌等多种人类恶性肿瘤密切相关。这一目标需要一个详细的机制理解的同型二聚体动物FAS如何进行7个连续的反应和通道底物的长链脂肪酸的生物合成。目前的信息学研究表明，FAS不仅由多个催化结构域组成，而且具有多种构象的高迁移率。这些特点，加上它的大尺寸，使FAS成为一个独特的有吸引力和具有挑战性的结构功能研究系统。为了迎接这一挑战，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已经成立。该团队将采用系统的方法，通过结合从各种技术获得的数据，以尽可能高的分辨率阐明FAS结构。由于FAS的多构象的存在，一种新的混合实验和计算改进程序将被迭代地采用，以合并不同分辨率的结构和计算信息学，以便以更详细的方式生成酶的三维结构。此外，在配体存在下，单个或串联功能域晶体结构的精确原子细节对于理解催化机制和未来抗癌治疗的合理设计将是无价的。具体目标是：1)通过一种新的计算细化程序，通过冷冻电镜（cryo-EM）最终确定FAS至10a的三维结构。2)通过x射线晶体学确定FAS催化/功能域的原子结构，单个或串联。3)通过抗体fab片段和/或金标来定位结构域的位置，并将结构域的晶体结构与完整酶的冷冻电镜图相匹配。4)整合所有的结构信息学、计算信息学和生化信息学，以推导出FAS导致多步催化合成长链脂肪酸的作用方式的分子基础。

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.