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工作，并可能导致以天然产物为基础的化合物文库组合合成的新策略。Clardy实验室最近的研究发现了几种天然产物，包括雄酰胺、催产素C和达二胺，它们是由缺乏传统的非核糖体多肽合成酶和聚酮合成缩合酶的基因簇编码的。相反，这些天然产物似乎是由与初级代谢催化剂同源的非规范缩合酶构建的。这一令人惊讶的发现意味着整个类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，有可能通过基于序列的搜索发现全新的天然产物类别。在这个项目中，我将重点研究一种非规范缩合催化剂，即泛亚凝聚体中达二胺生物合成的DdaF酶。DdaF之所以吸引人，不仅是因为它的成键活性，还因为它表现出不同寻常的底物混杂性，这可能导致化合物类似物库的产生。我提出三个具体目标。目的1：通过确定DdaF是否负责第一步或第二步成键来阐明DdaF在达二胺生物合成中的作用。目的2：研究DdaF的底物混杂性，作为表征其在组合生物合成中的潜在效用的手段。我将通过筛选底物类似物库来表征DdaF的底物耐受性的程度，这些底物类似物旨在探索DdaF接受的结构要求。目的3：通过结合反应物和/或产物求解晶体结构来研究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的比较结构分析和基于结构的新的DdaF同源物搜索的基础

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