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

细菌和真菌的巨酶聚酮合成酶（pks）合成许多救命的聚酮类药物，包括抗癌、杀菌、抗真菌和降胆固醇化合物，以及许多用于农业和畜牧业的化学品。这些巨大的蛋白质由一系列催化和载体蛋白结构域组成，它们协同产生具有高结构和立体化学复杂性的分子，这些特征与它们的生物活性密切相关。在学术界和生物技术领域，pks的基因工程是一种很有前途的方法，可以获得具有改进性能和增加价值的理想类似物。这样的策略可能被用来解决，例如，我们迫切需要新的化疗药物和药物来解决细菌抗生素耐药性。然而，由于我们对PKS结构生物学的了解不足，这种策略仍然受到阻碍。主要的科学挑战是确定这些巨大的多酶的三维结构，提供对域间相互作用的关键见解，这将支持更有效的PKS重组。为了解决这一知识差距，我们的四个国际德国/法国团队合作(德国汉诺威大学A. Kirschning和R. J. Cox小组（合作伙伴1和2）；K. J. Weissman小组，洛林大学，法国（合伙人3）和S. Spinelli/C.。Cambillau小组，法国马赛大学（合作伙伴4）)提出，通过两层方法表征细菌（trans-AT型）的两个模型模块化PKS以及完整的真菌，迭代PKS的一系列不同结构域组成的完整PKS模块：通过小角度x射线散射（SAXS）进行初步分析，然后通过电子显微镜（阴性染色和冷冻电镜）在更高分辨率下对选择的结构进行详细研究。目标蛋白将以载脂蛋白和holo（用假体磷酸蚁氨酸修饰）形式，以及天然中间体和化学合成的结构域交联剂的存在进行研究。因此，该项目依赖于四个具有高度互补性技能的实验室的密切合作，符合DFGs和ANRs培养强大的国际研究项目的目标。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有效性，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引入临床应用。

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.