Structural Biology of Complex Enzymes

描述(申请人提供)：复杂的酶在生物合成途径中执行多步催化，在生物合成途径中，不稳定或不溶解的中间体在活性部位之间进行通道或运输。我们建议继续我们对一个复杂的酶系统，谷氨酰胺氨基转移酶的结构研究，并开始第二个，咖喱酸聚酮合成酶的工作。谷氨酰胺氨基转移酶是一种生物合成酶，它从谷氨酰胺的一个活性部位产生氨，并将其引导到第二个活性部位，以添加到第二个底物。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合成酶的生物中催化tRNAGln或tRNAAsn氨酰化的后半部分。具体地说，我们将研究Aquifex aeolicus GatCAB与tRNA和底物样小分子的复合体。这些研究旨在了解GatB亚基是如何识别错误充电的Glu-tRNAGln或Asp-tRNAAsn的，tRNA结合如何刺激GATA亚基中的氨产生，氨是如何在亚基之间输送的，以及GatB中的氮的亲核加成产生Gln-tRNAGln或Asn-tRNAAsn的基于结构的机制是什么。在目标2和目标3中，我们将重点介绍一类新的复杂酶--模块化聚酮合成酶(PKSS)。微生物产生的聚酮类天然产物种类繁多，是新的生物活性分子的重要来源，可作为开发新药物的先导化合物。在这些巨型酶中，一个载体结构域在酶活性部位之间运输不溶的中间产物。我们的主题是库拉星A的PKS，这是一种具有抗微管蛋白活性的聚酮。我们将研究柯拉酸三个不寻常的化学特征的结构生物化学：环丙烷环、顺式双键和端烯。我们将产生由75kb库拉星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的晶体结构，并检测催化专一性。这些研究的结果将是了解咖喱素PKS是如何进行其不寻常的化学转化的，以及催化结构域可以容忍底物变体的程度。与公共健康相关：许多酶执行复杂的多步骤化学反应。这个项目将通过解决酶-底物或类似物的晶体结构来研究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域如何将反应产物引导到下一反应的位置。

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.