Engineered Biosynthesis of Novel Macrolactone Polyketides

描述(申请人提供)：6-脱氧赤藓内酯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，我们已经：(I)解决了两个DEBS模块主要部分的X-射线晶体结构；(Ii)解决了来自DEBS的一个原型酰基载体蛋白结构域的核磁共振结构；(Iii)研究了聚酮生物合成中关键步骤的机理方面，包括模块间的链转移、链延长、延伸单元的选择和转移、β-酮还原、脱水和大内酯化；(Iv)探索了蛋白质-蛋白质相互作用在建立总体结构和控制PKS模块的选择性中的作用；以及(V)探讨了分子内结构对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学含义也被探索和利用。在接下来的项目期间，我们建议继续对DEBS的结构、机理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)KS和ACP相互交联的Debs模块的片段；以及(Iii)独立的酰基转移酶(Dsz AT)，它在反式PKS模块的酰化方面非常有效。我们还将研究：(I)模块内和模块间酮合成酶-酰基载体蛋白的专一性；(Ii)Dsz AT的高效转酰化；(Iii)整个DEBS催化循环的速率控制，以及当非自然延伸单元加入时它是如何受到影响的。最后但并非最不重要的是，为了强调上述知识的实际意义，我们将：(I)阐述新发现的红霉素的构效关系，该关系可能导致在真核细菌核糖体的潜在新结合口袋内进行探索；以及(Ii)尝试确定一种新的先导物质，该先导物质具有抗顶复合体寄生虫的药用活性。与公共健康相关：负责红霉素大环核心生物合成的酶装配线是多模聚酮合成酶研究得最好的例子。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)对红霉素合成酶的分子逻辑有一个基本的了解；(Ii)利用这一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.