Understanding programming in highly reducing iterative fungal polyketide synthases - a structural and mechanistic approach

真菌聚酮合成酶(PKS)是一类超大型、多功能、多结构域的蛋白质，它以高度程序化的迭代方式将简单的生化构件组装在一起，形成一系列具有生物活性的复杂天然产物，其中许多是重要的药物或农用化学品，或者是对人类健康和农业都有害的毒素。简而言之，PKS就像一根绳子上的珠子，其中PKS的每个珠子或‘结构域’代表一个单独的酶功能或生物催化剂。PKS使用简单的碳-碳键形成(缩合)反应，然后是各种剪裁反应，在这些反应中，初始缩合产物可能会被一系列反应中的全部或部分改变-还原(添加两个氢原子)、脱水(失水)和C-甲基化(添加一个碳分支)，每个反应都由PKS上的特定结构域控制。发现控制这种编程的分子因素可以说是我们理解自然如何制造复杂的天然产物的最大遗留问题，也是酶催化的一个重大基本问题。对编程如何工作，如何确定总链长、还原程度和甲基化的数字迭代缩合周期的理解基本上是空白的。在这个项目中，将分离出单独的珠子/结构域，以便研究它们的单独属性和结构。然后，各个珠子将被聚集在一起，研究一个珠子如何影响另一个珠子，这个过程将重复进行，试图建立一个完整的PKS如何工作的总体图景。该项目是高度跨学科的，将汇集天然产物化学、化学合成、分子生物学、生物化学、结构生物学和生物信息学的技能和专业知识。其最终目的是利用对规划实际如何运作的了解，操纵PKSS来制造对人类和动物健康有益的改变或改进的特性的新产品，并为制药和精细化工行业提供生产新的有用产品的新方法。

Fungal polyketide synthases (PKS) are extremely large multifunctional multi-domain proteins that assemble simple biochemical building blocks in a highly programmed iterative manner to make a huge range of biologically active complex natural products many of which are of importance as drugs or agrochemical agents or as toxins which are detrimental to both human health and agriculture. In simple terms PKSs are like beads on a string where each bead or 'domain' of the PKS represents an individual enzyme function or biological catalyst. The PKS uses simple carbon-carbon bond forming (condensation) reactions followed by a variety of 'tailoring' reactions in which the initial condensation product may be modified by all or some of a series of reactions - reduction (addition of two atoms of hydrogen), dehydration (loss of water) and C-methylation (addition of a single carbon branch), each of which is controlled by specific domains on the PKS. Discovering the molecular factors which control this programming is arguably the greatest remaining problem in our understanding of how nature makes complex natural products, and also represents a great fundamental problem in enzyme catalysis. Understanding of how the programming works, how the number iterative condensation cycle that determines the overall chain length, degree of reduction, and methylation is essentially absent. In this project the individual beads/domains will be isolated so that their individual properties and structures can be studied. Individual beads will then be brought together to study how one influences another, and this process will be repeated to try to build up an overall picture of how the intact PKS works. The project is highly interdisciplinary and will bring together the skills and expertise of natural products chemistry, chemical synthesis, molecular biology, biochemistry, structural biology and bioinformatics. The ultimate aim is to use the understanding gained of how the programming actually works, to manipulate the PKSs to make new products with altered or improved properties of benefit to human and animal health and to provide the pharmaceutical and fine chemical industries with new methods to produce new useful products.