DdaF: A Novel Condensation Enzyme in Dapdiamide Biosynthesis

描述（由申请人提供）：抗生素耐药性是对人类健康的日益严重的威胁，需要开发新的有效治疗方法。天然产物已被证明是生物活性小分子的重要资源，但显然需要新的方法来加速它们的发现。对天然产物生物合成的深入了解将有助于天然产物的发现，并可能导致基于天然产物的化合物库的组合合成的新策略。克拉迪实验室最近的研究已经发现了几种天然产物，包括阿替米德、泛霉素C和dapdiamides，它们由缺乏非核糖体多肽合成酶和聚酮化合物合成的传统缩合酶的基因簇编码。相反，这些天然产物似乎是由与初级代谢催化剂同源的非典型缩合酶构建的。这一令人惊讶的发现意味着，整个类别的生物合成基因簇可能被错误地注释为属于初级代谢，而不是次级代谢。对这些新的成键催化剂及其合成逻辑的详细研究代表了一个令人兴奋的机会，可能会通过基于序列的搜索发现全新的天然产物。在这个项目中，我将集中在一个非典型的缩合催化剂，酶DdaF从达帕双酰胺生物合成的泛菌agglomerans。DdaF不仅因为其成键活性而有趣，而且还因为它表现出不寻常的底物混杂性，可以允许产生化合物类似物的文库。我提出三个具体目标。目标1：通过确定DdaF是否负责第一或第二键形成步骤来阐明DdaF在达帕双胺生物合成中的作用。目的2：研究DdaF的底物混杂性，以表征其在组合生物合成中的潜在用途。我将通过筛选一个旨在探测DdaF接受的结构要求的底物类似物库来表征DdaF的底物耐受性的程度。目的3：通过求解具有结合反应物和/或产物的晶体结构来研究DdaF的底物混杂性。DdaF的结构将作为与来自初级代谢的同源酶进行比较结构分析和基于结构搜索新的DdaF同系物的基础

DESCRIPTION (provided by applicant): Antibiotic resistance is a growing threat to human health, necessitating the development of new and effective therapeutics. Natural products have proven to be an important resource of biologically active small molecules, but new methods to hasten their discovery are clearly needed. A deeper understanding of natural product biosynthesis will aid natural product discovery efforts and potentially lead to new strategies for combinatorial synthesis of natural product-based compound libraries. Recent studies in the Clardy laboratory have uncovered several natural products, including andrimid, pantocin C, and the dapdiamides, that are encoded by gene clusters lacking the traditional condensation enzymes of non-ribosomal polypeptide synthetases and polyketide syntheses. Instead, these natural products appear to be constructed by non-canonical condensation enzymes with homology to catalysts from primary metabolism. This surprising discovery implies that entire classes of biosynthetic gene clusters may be misannotated as belonging to primary rather than secondary metabolism. Detailed study of these new bond-forming catalysts and their synthetic logic represents an exciting opportunity, potentially leading to the discovery of entirely new classes of natural products through sequence-based searching. In this project, I will focus on one non-canonical condensation catalyst, the enzyme DdaF from dapdiamide biosynthesis in Pantoea agglomerans. DdaF is intriguing not only for its bond-forming activity, but also because it demonstrates unusual substrate promiscuity that could allow for the production of libraries of compound analogues. I propose three specific aims. Aim 1: To elucidate the role of DdaF in dapdiamide biosynthesis by determining whether it is responsible for the first or the second bond-forming step. Aim 2: To investigate the substrate promiscuity of DdaF as a means of characterizing its potential utility for combinatorial biosynthesis. I will characterize the extent of DdaF's substrate tolerance by screening a library of substrate analogues designed to probe the structural requirements for acceptance by DdaF. Aim 3: To investigate the substrate promiscuity of DdaF by solving crystal structures with bound reactants and/or products. The structure of DdaF will serve as the basis for a comparative structural analysis with homologous enzymes from primary metabolism and for structure-based searching for novel DdaF homologues