Structural Biology of Complex Enzymes

复杂酶的结构生物学

• 批准号: 8003113
• 负责人: JANET L. SMITH
• 金额: $ 5.45万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003113
• 关键词: Active Sites; Address; Alkenes; Amination; Amino Acids; Amino Acyl-tRNA Synthetases; Amino Sugars; Aminoacylation; Ammonia; Asparagine-Specific tRNA; Asparagine-tRNA ligase; Binding; Biochemical; Biochemistry; Biological Factors; Carboxy-Lyases; Carrier Proteins; Catalysis; Catalytic Domain; Charge; Chemicals; Complex; Coupling; Covalent Interaction; Curacin A; Curacin Compound; Cyclopropanes; Development; Elements; Engineering; Enzymes; Gene Cluster; Generations; Glutaminase; Glutamine; Glutamine-Specific tRNA; Goals; Hydro-Lyases; Hydrolysis; Lead; Microbe; Multienzyme Complexes; Nature; Nitrogen; Nucleotides; Organism; Oxidoreductase; Pathway interactions; Pharmacologic Substance; Production; Proteins; Reaction; Side; Site; Solubility; Source; Specificity; Structural Biochemistry; Structure; Substrate Interaction; System; Tertiary Protein Structure; Transfer RNA; Transfer RNA Aminoacylation; Tubulin; Variant; Work; analog; base; chemical group; chemical reaction; cyclopropane; enzyme substrate; glutamine-tRNA; nucleophilic addition; pi bond; polyketide synthase; public health relevance; small molecule; structural biology

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.

描述（由申请人提供）：复杂的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。我们建议继续我们对一种复杂酶系统，谷氨酰胺氨基转移酶的结构研究，并启动第二次库拉蛋白聚酮化合物合酶的工作。谷氨酰胺酰胺氨基转移酶是生物合成酶，可在一个活性位点产生氨基氨基，并将其引导到第二个活性位点以添加第二个底物。在AIM 1中，我们将继续研究GATCAB，该研究催化缺乏谷氨酰胺TRNA合成酶或天冬酰胺TRNA合成酶的生物体中TRNAGLN或TRNAASN氨基酰化的下半年。具体而言，我们将检查具有tRNA和底物样小分子的aquifex aeolicus gatcab的复合物。 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.在AIMS 2和3中，我们将重点关注一类新的复杂酶，即模块化聚酮化合物合酶（PKSS）。微生物产生了惊人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，可以用作开发新药物的铅化合物。在这些大型酶中，载体结构域在酶活性位点之间运输不溶性中间体。我们的受试者是curacin a的PK，是具有抗微管蛋白活性的聚酮化合物。我们将研究素蛋白的三种异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75千洛碱库酸蛋白基因簇编码的相关催化结构域，确定域及其底物或模拟复合物的晶体结构，并检查催化特异性。这些研究的结果将是了解库氨蛋白PK如何进行其异常化学转化，以及催化域在多大程度上可以耐受底物变体。公共卫生相关性：许多酶执行复杂的多步化学反应。该项目将通过求解酶 - 底物或-Analog复合物的晶体结构来研究与多种复杂酶的底物相互作用的细节。目的是了解蛋白质的催化域如何将反应产物引导到下一个反应的位点。