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形成通道或转运。我们建议继续我们的结构研究的一个复杂的酶系统，谷氨酰胺酰胺转移酶，并开始工作的第二个，curacin聚酮合酶。谷氨酰胺酰胺转移酶是生物合成酶，其在一个活性位点从谷氨酰胺产生氨，并将其引导至第二活性位点以添加至第二底物。在目标1中，我们将继续研究GatCAB，其在缺乏谷氨酰胺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，我们将研究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亚基如何识别错误电荷的Glu-tRNAGln或Asp-tRNAAsn，tRNA结合如何刺激加塔亚基中的氨产生，氨如何在亚基之间传递，以及GatB中氮的亲核加成产生Gln-tRNAGln或Asn-tRNAAsn的结构机制是什么。在目标2和3中，我们将集中于一类新的复合酶，模块化聚酮酶（PKS）。微生物产生的聚酮化合物天然产物种类繁多，是新的生物活性分子的重要来源，可作为开发新药的先导化合物。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，一种具有抗微管蛋白活性的聚酮化合物的PKS。我们将研究结构生物化学的三个不寻常的化学特征：一个环丙烷环，一个顺式双键，和一个终端烯烃。我们将产生相关的催化结构域编码的75-脱氢酶curacin基因簇，确定晶体结构域和他们的底物或类似物的复合物，并检查催化特异性。这些研究的结果将是一个了解如何curacin PKS进行其不寻常的化学转化，以及在何种程度上的催化结构域可以容忍底物的变化。公共卫生相关性：许多酶进行复杂的多步化学反应。本计画将借由解析酶-底物或-类似物复合物的晶体结构，来探讨底物与几种复合酶相互作用的细节。目的是了解蛋白质的催化结构域如何将反应产物引导到下一个反应的位点。

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.