Structural basis of specificity in affinity-labeled polyketide synthase didomains

亲和标记聚酮合酶双结构域特异性的结构基础

• 批准号: 7744652
• 负责人: JANET L. SMITH
• 金额: $ 48.68万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7744652
• 关键词: Active Sites; Acyl Carrier Protein; Address; Affinity Labels; Anabolism; Antibiotics; Bacteria; Bacterial Drug Resistance; Binding; Biochemistry; Biological Assay; Biological Factors; Carbon; Catalytic Domain; Chemicals; Clinical; Collaborations; Complex; Crystallography; Engineering; Enzymes; Erythromycin; Face; Genetic Recombination; Goals; Ketones; Knowledge; Label; Lead; Length; Lovastatin; Macrolide Antibiotics; Methods; Multi-Drug Resistance; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Positioning Attribute; Probability; Protein Engineering; Public Health; Publications; Reaction; Research; Research Design; Sirolimus; Source; Specificity; Structural Biologist; Structure; Substrate Domain; Substrate Interaction; Substrate Specificity; Synthesis Chemistry; System; Tertiary Protein Structure; Testing; Tylosin; Water; Work; adduct; affinity labeling; analog; base; catalyst; combat; combinatorial; design; drug discovery; fascinate; interest; novel; picromycin; polyketide synthase; public health relevance; research study; restraint; stereochemistry; thioether; tool

DESCRIPTION (provided by applicant): Acquired resistance to antibacterial drugs is a growing public health problem due to the spread of multidrug-resistant bacteria. The long-term objective of this project is to build the knowledge to engineer new combinations of polyketide synthase (PKS) catalytic domains with reliable predictions of the chemical and stereochemical outcome. Modular PKSs are among the most desirable target pathways for "combinatorial biosynthesis" because of the rich chemical diversity of their products. They are also among the most amenable pathways to this approach because of their modular organization. Recombination of PKS domains to build synthetic pathways for novel compounds requires an understanding of the basis for substrate specificity and stereochemical outcome for each catalytic domain, which at present is lacking. Crystal structures of some domains are available, but, apart from the preliminary studies for this application, the structures lack bound substrates or analogs. Affinity labels have been demonstrated to be an effective means to establish the mechanism of pikromycin thioesterase (Pik TE). The central hypothesis of the proposed research is that polyketide-based affinity labels are powerful tools to probe the structure and mechanism of isolated PKS didomains. The specific aims of this application are: (1) to determine the structural basis for substrate specificity and stereochemical outcome of several NADP-dependent ketoreductase (KR) domains, (2) to explore the substrate range of KR domains for non-natural substrates, and (3) to understand the mechanisms of macrolactone formation by terminal thioesterase (TE) domains. The research design and methods will utilize the acyl carrier protein (ACP) of didomain constructs, KR-ACP and ACP-TE, to deliver substrate and product mimics to the KR and TE active sites. Natural KR-ACP and ACP-TE didomains will be covalently modified by vinyl ketone affinity label mimics, and their crystal structures determined. Didomains from the PKS systems for pikromycin, erythromycin, and tylosin will be examined since these molecules are established lead compounds for antibiotic drug discovery. The proposed research is significant because the rational engineering of PKS systems is expected to provide novel macrolide antibiotics to combat the rising tide of multidrug-resistant bacteria. PUBLIC HEALTH RELEVANCE: Complex mega-enzyme machines known as polyketide synthases (PKS) produce many antibiotics and other bio-active molecules. With appropriate engineering, these proteins are potentially rich sources of new pharmaceuticals. This project will examine the details of substrate interactions with several PKS domains. Substrate mimics will be synthesized that covalently attach to the enzyme, and crystal structures of substrate mimic trapped in the enzyme will be solved. The goal is to understand how the substrate specificity and range for each reaction, and thereby allow design of new enzymes to make new bio-active molecules.

描述(申请人提供)：由于多重耐药细菌的传播，对抗菌药物的获得性耐药性是一个日益严重的公共卫生问题。该项目的长期目标是建立知识来设计新的聚酮合成酶(PKS)催化结构域组合，并可靠地预测化学和立体化学结果。模块化PKS由于其产物丰富的化学多样性而成为“组合生物合成”最理想的靶向途径之一。由于它们的模块化组织，它们也是这种方法最容易接受的途径之一。重组PKS结构域以建立新化合物的合成途径需要了解底物专一性的基础和每个催化域的立体化学结果，这是目前所缺乏的。一些结构域的晶体结构是可用的，但除了对这一应用的初步研究外，这些结构缺乏结合底物或类似物。亲和标记已被证明是建立吡克罗霉素硫代酯酶(Pik TE)作用机制的有效手段。这项研究的中心假设是，基于聚酮的亲和标记是探索分离的PKS双域结构和机制的有力工具。本应用的具体目的是：(1)确定几个依赖于NADP的酮还原酶(KR)结构域的底物专一性和立体化学结果的结构基础；(2)探索非天然底物的KR结构域的底物范围；(3)了解末端硫酯酶(TE)结构域形成大内酯的机制。研究设计和方法将利用双域结构的酰基载体蛋白(ACP)，KR-ACP和ACP-TE，将底物和产物模拟物输送到KR和TE活性部位。天然的KR-ACP和ACP-TE双域将被乙烯基酮亲和标记模拟物共价修饰，并测定了它们的晶体结构。由于吡克罗霉素、红霉素和泰乐菌素是抗生素药物发现的先导化合物，因此将研究这些分子在PKS系统中的作用。这项拟议的研究具有重要意义，因为合理设计PKS系统有望提供新的大环内酯类抗生素，以对抗日益高涨的多重耐药细菌。与公共健康相关：被称为聚酮合成酶(PKS)的复杂巨酶机器会产生许多抗生素和其他生物活性分子。通过适当的工程，这些蛋白质可能是新药物的丰富来源。本项目将研究底物与几个PKS结构域相互作用的细节。将合成底物模拟物，将其共价连接到酶上，并解决捕获在酶中的底物模拟物的晶体结构。我们的目标是了解底物如何针对每个反应的专一性和范围，从而允许设计新的酶来制造新的生物活性分子。