Engineered Biosynthesis of Novel Macrolactone Polyketides

描述（由申请人提供）：6-脱氧乌洛托品B合酶（DEBS）是研究最广泛的多模块聚酮合酶（PKS）。在过去的项目期间，我们已经：（i）解决了两个DEBS模块的主要部分的X射线晶体结构;（ii）解决了DEBS的原型酰基载体蛋白结构域的NMR结构;（iii）研究了聚酮生物合成中关键步骤的机理，包括模块间链转移，链延伸，延伸单元的选择和转移，？酮还原，脱水和macrolactonization;（iv）探讨了蛋白质-蛋白质相互作用的作用，建立整体架构和控制的PKS模块的选择性;和（v）询问的DEBS催化循环内的个别反应的速率的影响intramolecularity。这些和其他见解的生物合成工程的影响也被探索和利用。在下一个项目期间，我们建议继续对DEBS的结构，机制和工程进行基础和应用研究。具体地说，我们将试图解决三个新的蛋白质构建体的X射线晶体结构，包括：（i）由几乎完整的DEBS模块组成的大片段;（ii）其中KS和ACP已经相互交联的DEBS模块片段;和（iii）独立酰基转移酶（Dsz AT）我们还将研究以下机制基础：（i）模块内和模块间酮合酶-酰基载体蛋白特异性;（ii）通过Dsz AT的有效转酰作用;和（iii）整个DEBS催化循环的速率控制，以及当引入非天然的延伸剂单元时它如何受到影响。最后但并非最不重要的是，为了突出上述知识的实际相关性，我们将：（i）阐述红霉素新发现的结构-活性关系，这可能导致在真细菌核糖体的潜在新结合口袋内的探索;和（ii）尝试鉴定具有针对顶复门寄生虫的医学相关活性的新型先导物质。公共卫生相关性：负责红霉素大环核心生物合成的酶组装线是多模块聚酮合酶的最佳研究实例。这个酶家族在自然界中产生许多重要的抗生素。该项目的两个主要目标是：（i）获得红霉素合酶的分子逻辑的基本理解;和（ii）利用这些知识来制造新的抗生素。

DESCRIPTION (provided by applicant): The 6-deoxyerythronolide B synthase (DEBS) is the most extensively studied multimodular polyketide synthase (PKS). During the past project period, we have: (i) solved the X-ray crystal structures of major portions of two DEBS modules; (ii) solved the NMR structure of a prototypical acyl carrier protein domain from DEBS; (iii) investigated mechanistic aspects of key steps in polyketide biosynthesis including intermodular chain transfer, chain elongation, extender unit selection and transfer, ?-ketoreduction, dehydration and macrolactonization; (iv) explored the role of protein-protein interactions in establishing the overall architecture and controlling the selectivity of a PKS module; and (v) interrogated the effect of intramolecularity on the rates of individual reactions within the DEBS catalytic cycle. The biosynthetic engineering implications of these and other insights have also been explored and exploited. During the next project period, we propose to continue our fundamental and applied studies on the structure, mechanism and engineering of DEBS. Specifically, we will attempt to solve the X-ray crystal structures of three new protein constructs including: (i) a large fragment consisting of a nearly complete DEBS module; (ii) a fragment of a DEBS module in which the KS and ACP have been crosslinked to each other; and (iii) a stand-alone acyl transferase (Dsz AT) that is exceptionally efficient at acylating PKS modules in trans. We will also investigate the mechanistic basis for: (i) intra- and inter- modular ketosynthase - acyl carrier protein specificity; (ii) efficient transacylation by Dsz AT; and (iii) rate control of the overall DEBS catalytic cycle, and how it is influenced when unnatural extender units are incorporated. Last but not least, to highlight the practical relevance of the above knowledge, we will: (i) elaborate a newly discovered structure-activity relationship for erythromycin that could lead to explorations within a potentially new binding pocket of the eubacterial ribosome; and (ii) attempt to identify a novel lead substance with medicinally relevant activity against apicomplexan parasites. PUBLIC HEALTH RELEVANCE: The enzymatic assembly line responsible for the biosynthesis of the macrocyclic core of erythromycin is the best-studied example of a multimodular polyketide synthase. This enzyme family produces many important antibiotics in nature. The two principal goals of this project are: (i) to obtain a fundamental understanding of the molecular logic of the erythromycin synthase; and (ii) to exploit this knowledge to make new antibiotics.