PKS_STRUCTURE - Structural characterization of drug-producing polyketide synthase multienzymes by electron microscopy, small-angle X-ray scattering and allied biophysical and synthetic chemistry approaches

PKS_STRUCTURE - 通过电子显微镜、小角 X 射线散射以及相关生物物理和合成化学方法对药物生产聚酮合酶多酶进行结构表征

• 批准号: 316629434
• 负责人: Professor Dr. Russell J. Cox
• 金额: --
• 依托单位: Institut für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/316629434?language=en
• 关键词: PKS_STRUCTURE; Structural; characterization; drug; producing

The mega-enzyme polyketide synthases (PKSs) of bacteria and fungi synthesize many life-saving polyketide medicines, including anticancer, bactericidal, anti-fungal and cholesterol-lowering compounds, as well as numerous chemicals used in agriculture and animal husbandry. These huge proteins are composed of a series of catalytic and carrier protein domains, which cooperate to generate molecules of high structural and stereochemical complexity- features which are intimately linked to their bioactivities. Genetic engineering of PKSs is a promising approach in both academia and the biotechnology sector to obtain desirable analogues with improved properties and increased value. Such a strategy might be used to address, for example, our urgent need for novel chemotherapeutics and agents to tackle bacterial antibiotic resistance. However, this strategy remains hampered by our insufficient understanding of PKS structural biology. The major scientific challenge is to determine the three-dimensional architectures of these gigantic multienzymes, providing the key insights into inter-domain interactions that will underpin more efficient PKS re-engineering. To address this knowledge gap, our four-team international German/French collaboration (A. Kirschning and R. J. Cox groups, University of Hannover, Germany (Partners 1 and 2); K. J. Weissman group, University of Lorraine, France (Partner 3) and S. Spinelli/C. Cambillau group, University of Marseille, France (Partner 4)) proposes to characterize a range of intact PKS modules of varying domain composition from two model modular PKSs of bacteria (trans-AT type), as well as an intact fungal, iterative PKS by a two-tiered approach: initial analysis by small-angle X-ray scattering (SAXS), followed by detailed investigation of select constructs at higher resolution by electron microscopy (both negative-staining and cryo-EM). The target proteins will be investigated in their apo and holo (modified with the prosthetic group phosphopantetheine) forms, as well as in the presence of native intermediates and domain cross-linking agents prepared by chemical synthesis. The project thus depends on the close collaboration of four laboratories with highly complementary skills, in line with the DFGs and ANRs objective of fostering strong, international research programs. The major scientific result will be to reveal the overall architectures of multiple PKS multienzymes, as well as large-scale conformational changes related to the functional states of the proteins. The data we obtain will dramatically increase the efficacy of synthetic biology experiments aimed at re-engineering PKS machineries towards the generation of new compounds with therapeutic potential. This project will thus further our ultimate goal of bringing engineering-derived polyketide analogues into clinical use via collaborations with pharmaceutical and/or biotechnology companies.

细菌和真菌的巨酶聚酮酶（PKS）合成许多挽救生命的聚酮药物，包括抗癌、杀菌、抗真菌和降低胆固醇的化合物，以及用于农业和畜牧业的许多化学品。这些巨大的蛋白质由一系列催化和载体蛋白结构域组成，这些结构域合作产生高度结构和立体化学复杂性的分子-这些特征与其生物活性密切相关。PKS的基因工程在学术界和生物技术领域都是一种很有前途的方法，可以获得具有改进性质和增加价值的理想类似物。例如，这种策略可以用于解决我们对新型化疗药物和药物的迫切需求，以解决细菌抗生素耐药性。然而，这一战略仍然阻碍了我们的PKS结构生物学的理解不足。主要的科学挑战是确定这些巨大的多酶的三维结构，提供关键的见解域间的相互作用，将支持更有效的PKS再工程。为了解决这一知识差距，我们的四个团队的国际德国/法国合作（A。Kirschning和R.德国汉诺威大学J.考克斯小组（合作伙伴1和2）;法国洛林大学J. Weissman小组（合作伙伴3）和S.斯皮内利角法国马赛大学Cambillau小组（合作伙伴4））提出从细菌的两个模型模块化PKS中表征一系列不同结构域组成的完整PKS模块（反式AT型），以及完整的真菌，迭代PKS的两层方法：通过小角X射线散射（SAXS）进行初步分析，随后通过电子显微镜（负染色和冷冻-EM）以更高的分辨率详细研究选择的构建体。靶蛋白将以其apo和holo（用辅基磷酸泛酰巯基乙胺修饰）形式以及存在天然中间体和通过化学合成制备的结构域交联剂的情况下进行研究。因此，该项目依赖于四个具有高度互补技能的实验室的密切合作，符合DFG和ANR促进强大的国际研究计划的目标。主要的科学成果将是揭示多个PKS多酶的整体结构，以及与蛋白质功能状态相关的大规模构象变化。我们获得的数据将大大提高合成生物学实验的效率，这些实验旨在重新设计PKS机制，以产生具有治疗潜力的新化合物。因此，该项目将进一步实现我们的最终目标，即通过与制药和/或生物技术公司的合作，将工程衍生的聚酮类似物投入临床使用。