DdaF: A Novel Condensation Enzyme in Dapdiamide Biosynthesis

描述(申请人提供)：抗生素耐药性对人类健康的威胁越来越大，需要开发新的有效疗法。天然产物已被证明是生物活性小分子的重要来源，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，并可能导致基于天然产物的化合物文库的组合合成的新策略。克拉迪实验室最近的研究发现了几种天然产物，包括雄胺、泛素C和达苯二胺，它们是由缺乏非核糖体多肽合成酶和聚酮合成的传统缩合酶的基因簇编码的。相反，这些天然产物似乎是由非典型缩合酶构成的，与初级代谢的催化剂具有同源性。这一令人惊讶的发现意味着，生物合成基因簇的整个类别可能被错误地解释为属于初级新陈代谢，而不是次级新陈代谢。对这些新的成键催化剂及其合成逻辑的详细研究代表着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品类别。在这个项目中，我将重点研究一种非典型的缩合催化剂--聚集泛亚生物合成达苯二胺的酶DDAF。DDAF之所以耐人寻味，不仅是因为它的成键活性，还因为它展示了不寻常的底物混杂，这可能允许生产化合物类似物的库。我提出了三个具体目标。目的1：通过确定DDAF在第一步或第二步成键过程中的作用，阐明DDAF在氨苯二胺生物合成中的作用。目的：研究DDAF的底物杂交性，以表征其在组合生物合成中的潜在用途。我将通过筛选底物类似物文库来表征DDAF底物耐受性的程度，该文库旨在探索DDAF接受底物的结构要求。目的3：通过结合反应物和/或产物的晶体结构来研究DDAF的底物混杂。DDAF的结构将作为与初级代谢中的同源酶进行比较结构分析以及基于结构搜索新的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