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（由申请人提供）：由于多药耐药细菌的传播，对抗菌药物的抗药性是日益增长的公共卫生问题。该项目的长期目的是建立知识，以设计聚酮化合酶（PKS）催化域的新组合，并可靠地预测化学和立体化学结果。模块化PKS是“组合生物合成”的最理想的目标途径之一，因为其产品的化学多样性丰富。由于其模块化组织，它们也是这种方法最适合的途径之一。 PKS结构域的重组以建立新化合物的合成途径，需要了解每个催化域的底物特异性和立体化学结果的基础，目前缺乏该催化域。某些结构域的晶体结构可用，但是除了该应用的初步研究外，结构还缺乏结合的底物或类似物。亲和力标签已被证明是建立长霉素硫酯酶（PIK TE）机制的有效手段。拟议的研究的中心假设是，基于聚酮化合物的亲和力标签是探测孤立PKSDOMAIN的结构和机制的强大工具。该应用的具体目的是：（1）确定几个依赖NADP依赖性酮核酶（KR）域（2）的底物特异性和立体化学结果的结构基础，以探索非天然基质的KR域的底物范围，以及（3）通过末端thioestersane（TETIOSESTERSAPES）的机制（TETE）的机制（3）。研究设计和方法将利用DDOMAN构建体的酰基载体蛋白（ACP），KR-ACP和ACP-TE，将底物和产品模仿传递到KR和TE活性位点。天然的KR-ACP和ACP-TE DEDOMAIN将通过乙烯基酮亲和力标签模拟物及其晶体结构来共价修饰。 PKRomycin，红霉素和泰糖苷的PKS系统的散发蛋白因素将被检查，因为这些分子是建立抗生素药物发现的铅化合物。拟议的研究之所以重要，是因为PKS系统的合理工程有望提供新型的大花环抗生素来应对多药耐药细菌的上升潮流。公共卫生相关性：称为Polyketide合酶（PK）的复杂巨型酶机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的潜在丰富来源。该项目将检查与多个PKS域的底物相互作用的细节。将合成底物模拟物，即共价附着在酶上，并且将求解被困在酶中的底物的晶体结构。目的是了解每个反应的底物特异性和范围，从而允许设计新酶制造新的生物活性分子。