Structural Biology of Complex Enzymes

描述（由申请人提供）：复合酶在生物合成途径中进行多步催化，其中不稳定或不溶性中间体在活性位点之间引导或运输。我们建议继续对一种复杂的酶系统（谷氨酰胺酰胺转移酶）进行结构研究，并开始研究第二种酶（curacin 聚酮合酶）。谷氨酰胺酰胺转移酶是一种生物合成酶，其从一个活性位点的谷氨酰胺产生氨并将其引导至第二个活性位点以添加到第二底物中。在目标 1 中，我们将继续研究 GatCAB，它在缺乏谷氨酰胺 tRNA 合成酶或天冬酰胺 tRNA 合成酶的生物体中催化 tRNAGln 或 tRNAAsn 氨酰化的后半部分。具体来说，我们将检查 Aquifex aeolicus GatCAB 与 tRNA 和底物样小分子的复合物。这些研究旨在了解 GatB 亚基如何识别错误带电的 Glu-tRNAGln 或 Asp-tRNAAsn、tRNA 结合如何刺激 GatA 亚基中氨的产生、氨如何在亚基之间传导，以及 GatB 中氮亲核加成产生 Gln-tRNAGln 或 Asn-tRNAAsn 的基于结构的机制是什么。在目标 2 和 3 中，我们将重点关注一类新型复杂酶，即模块化聚酮合酶 (PKS)。微生物产生种类繁多的聚酮化合物天然产物，它们是新生物活性分子的重要来源，可用作开发新药物的先导化合物。在这些巨型酶中，载体结构域在酶活性位点之间运输不溶性中间体。我们的主题是 curacin A 的 PKS，这是一种具有抗微管蛋白活性的聚酮化合物。我们将研究 Curacin 的三个不寻常化学特征的结构生物化学：环丙烷环、顺式双键和末端烯烃。我们将产生由 75-kilobase curacin 基因簇编码的相关催化结构域，确定结构域及其底物或类似物复合物的晶体结构，并检查催化特异性。这些研究的结果将是了解 Curacin PKS 如何进行其不寻常的化学转化，以及催化结构域在多大程度上可以耐受底物变异。公共卫生相关性：许多酶会进行复杂的多步骤化学反应。该项目将通过解析酶-底物或类似物复合物的晶体结构来检查底物与几种复杂酶相互作用的细节。目标是了解蛋白质的催化结构域如何将反应产物引导至下一个反应位点。

DESCRIPTION (provided by applicant): Complex enzymes perform multi-step catalysis in biosynthetic pathways in which labile or insoluble intermediates are channeled or transported between active sites. We propose to continue our structural studies of one complex enzyme system, the glutamine amidotransferases, and to initiate work on a second, the curacin polyketide synthase. The glutamine amidotransferases are biosynthetic enzymes that generate ammonia from glutamine in one active site and channel it to a second active site for addition to a second substrate. In Aim 1, we will continue studies of GatCAB, which catalyzes the second half of tRNAGln or tRNAAsn aminoacylation in organisms lacking a glutamine tRNA synthetase or asparagine tRNA synthetase. Specifically, we will examine complexes of Aquifex aeolicus GatCAB with tRNA and substrate-like small molecules. These studies aim to understand how the GatB subunit recognizes mis-charged Glu-tRNAGln or Asp-tRNAAsn, how tRNA binding stimulates ammonia production in the GatA subunit, how ammonia is channeled between subunits, and what is the structure-based mechanism of nucleophilic addition of nitrogen in GatB to produce Gln-tRNAGln or Asn-tRNAAsn. In Aims 2 and 3, we will focus on a new class of complex enzymes, the modular polyketide synthases (PKSs). Microbes produce an astounding variety of polyketide natural products, which are an important source of new bio-active molecules that can be exploited as lead compounds for development of new pharmaceuticals. In these mega-enzymes, a carrier domain transports insoluble intermediates between enzyme active sites. Our subject is the PKS for curacin A, a polyketide with anti-tubulin activity. We will study the structural biochemistry of three unusual chemical features of curacin: a cyclopropane ring, a cis double bond, and a terminal alkene. We will produce relevant catalytic domains encoded by the 75-kilobase curacin gene cluster, determine crystal structures of the domains and of their substrate or analog complexes, and examine catalytic specificity. The result of these studies will be an understanding of how the curacin PKS carries out its unusual chemical transformations, and to what extent the catalytic domains can tolerate substrate variants. PUBLIC HEALTH RELEVANCE: Many enzymes perform complex multi-step chemical reactions. This project will examine the details of substrate interactions with several complex enzymes by solving crystal structures of enzyme-substrate or -analog complexes. The goal is to understand how catalytic domains of proteins channel a reaction product to the site of the next reaction.