Engineered Biosynthesis of Novel Macrolactone Polyketides

描述（由申请人提供）：6-脱氧红素内酯B合成酶（DEBS）是研究最广泛的多模聚酮合成酶（PKS）。在过去的项目期间，我们解决了两个DEBS模块主要部分的x射线晶体结构；（ii）从DEBS中求解了一个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关键步骤的机理方面，包括模间链转移，链延伸，扩展剂单元选择和转移，？-酮还原、脱水和大内酯化；（iv）探索了蛋白-蛋白相互作用在建立PKS模块的整体结构和控制其选择性中的作用；(v)探究分子内分子对DEBS催化循环中单个反应速率的影响。这些和其他见解的生物合成工程含义也被探索和利用。在下一个项目期间，我们将继续在DEBS的结构、机理和工程方面进行基础和应用研究。具体来说，我们将尝试解决三种新蛋白质结构的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）一个由KS和ACP相互交联的DEBS模块的片段；（iii）一种独立的酰基转移酶（Dsz AT），在trans中酰基化PKS模块方面非常有效。我们还将研究酮合酶-酰基载体蛋白特异性的机制基础：(i)模块内和模块间；（ii） Dsz AT的高效转酰化；（iii）整个DEBS催化循环的速率控制，以及当加入非自然扩展剂单元时对其的影响。最后但并非最不重要的是，为了强调上述知识的实际相关性，我们将：(i)阐述新发现的红霉素的结构-活性关系，这可能导致探索真细菌核糖体的潜在新结合袋；（ii）尝试鉴定一种具有抗顶复合体寄生虫医学相关活性的新型铅物质。公共卫生相关性：负责红霉素大环核心生物合成的酶装配线是多模块聚酮合成酶研究得最好的例子。这种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)获得对红霉素合酶分子逻辑的基本理解；(2)利用这些知识制造新的抗生素。

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.