Structure and Mechanism in the Tautomerase Superfamily

互变酶超家族的结构和机制

• 批准号: 7589405
• 负责人: CHRISTIAN P. WHITMAN
• 金额: $ 27.91万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589405
• 关键词: 4-oxalocrotonate tautomerase; Acids; Active Sites; Address; Amino Acids; Antibiotic Resistance; Bacteria; Bioinformatics; Bioremediations; Coenzymes; Development; Drug resistance; Enzymatic Biochemistry; Enzymes; Evolution; Family; Funding; Generations; Goals; HIV; Homologous Protein; Hydrolysis; Ions; Isomerase; Isomerism; Kinetics; Lactams; Lead; Link; Metabolism; Metals; Migration Inhibitory Factor; Modeling; Molecular Biology; Mutation; Mycobacterium tuberculosis; Nature; Organism; Pharmaceutical Preparations; Phylogenetic Analysis; Physiological; Prevalence; Principal Investigator; Process; Proline; Public Health; Resistance; Resources; Role; Role playing therapy; Route; Staging; Structure; Structure-Activity Relationship; System; Title; Work; X-Ray Crystallography; base; catalyst; cis-3-chloroacrylic acid dehalogenase; design; enzyme mechanism; enzyme model; insight; malonate semialdehyde decarboxylase; member; monomer; phenylpyruvate tautomerase; programs; resistance mechanism; trans-3-chloroacrylic acid dehalogenase

DESCRIPTION (provided by applicant): The tautomerase superfamily, a group of structurally homologous proteins that share a common building block (the 2-1-2 motif) and a catalytic amino-terminal proline (Pro-1), is rich in mechanistic, structural, and evolutionary questions. The long-term goal of this project is to address these questions by a combination of mechanistic enzymology, molecular biology, X-ray crystallography, and bioinformatics. In the last funding period, mechanisms and structures were established for three superfamily members [the isomer-specific 3-chloroacrylic acid dehalogenases, designated CaaD and cis-CaaD, and malonate semialdehyde decarboxylase (MSAD)], showing how Nature used the 2-1-2 motif to create structural and mechanistic diversity. The stage is now set to use these enzymes as experimental vehicles to address fundamental questions about how enzymes work, how they evolve, and how new activities arise. The proposed studies will identify the underlying principles used in this system so that we might ultimately mimic Nature's processes and create new activities and structures using the 2-1-2 motif. Our major specific aims will be to (1) establish substrate orientation and interactions in the three active sites; (2) delineate the consequences of key mutations in CaaD, cis-CaaD, and MSAD; (3) carry out a pre-steady state kinetic analysis of CaaD and cis-CaaD; and (4) examine the role of catalytic promiscuity in the evolution of the tautomerase superfamily and establish evolutionary relationships by phylogenetic and bioinformatics analysis. The results set the stage for the generation of enzymatic activities using the 2-1-2 template. These studies will enhance our understanding of enzyme mechanisms and bacterial metabolism, lead to a better understanding of the role played by catalytic promiscuity in divergent evolution, and assist in the design of environmentally friendly proline-based biocatalysts. It is critical to understand how enzymes evolve due to the prevalence of antibiotic-resistant bacteria and other drug-resistant organisms (e.g., M. tuberculosis and HIV). One mechanism for resistance involves the enzymatic inactivation of a drug (e.g., 2-lactam hydrolysis). Resistance enzymes can evolve by amplification of a low-level resistance activity in a physiological enzyme. Thus, a well-defined model for divergent evolution is a valuable resource for understanding how resistance activities evolve in the first place and could suggest more effective strategies for overcoming drug-resistant organisms. PUBLIC HEALTH RELEVANCE: It is critical to understand how enzymes evolve and acquire new functions due to the prevalence of antibiotic-resistant bacteria and other drug-resistant organisms such as M. tuberculosis and HIV. Drug-resistant organisms have become a major public health threat and will continue to be one. The proposed studies will result in a well-defined model for the evolution of enzymes and enhance our understanding of how resistance evolves in the first place.

描述（由申请人提供）：互变异构酶超家族是一组结构同源的蛋白质，具有共同的构建模块（2-1-2 基序）和催化氨基末端脯氨酸 (Pro-1)，富含机械、结构和进化问题。该项目的长期目标是通过结合机械酶学、分子生物学、X 射线晶体学和生物信息学来解决这些问题。在上一个资助期间，为三个超家族成员[异构体特异性3-氯丙烯酸脱卤酶，命名为CaaD和顺式CaaD，以及丙二酸半醛脱羧酶（MSAD）]建立了机制和结构，展示了Nature如何使用2-1 -2 创造结构和机制多样性的主题。现在准备使用这些酶作为实验工具来解决有关酶如何工作、如何进化以及如何产生新活性的基本问题。拟议的研究将确定该系统中使用的基本原理，以便我们最终可以模仿自然的过程并使用 2-1-2 主题创建新的活动和结构。我们的主要具体目标是（1）在三个活性位点建立底物方向和相互作用； (2) 描述 CaaD、cis-CaaD 和 MSAD 关键突变的后果； (3)进行CaaD和cis-CaaD的预稳态动力学分析； (4) 研究催化混杂在互变异构酶超家族进化中的作用，并通过系统发育和生物信息学分析建立进化关系。结果为使用 2-1-2 模板产生酶活性奠定了基础。这些研究将增强我们对酶机制和细菌代谢的理解，更好地理解催化混杂在趋异进化中所发挥的作用，并有助于设计环保的脯氨酸基生物催化剂。了解酶如何因抗生素耐药细菌和其他耐药生物（例如结核分枝杆菌和艾滋病毒）的流行而进化至关重要。耐药性的一种机制涉及药物的酶促失活（例如 2-内酰胺水解）。抗性酶可以通过放大生理酶中的低水平抗性活性而进化。因此，一个明确的趋异进化模型是理解耐药性活动如何进化的宝贵资源，并且可以提出更有效的克服耐药生物体的策略。公共卫生相关性：由于抗生素耐药细菌和其他耐药微生物（例如结核分枝杆菌和艾滋病毒）的普遍存在，了解酶如何进化并获得新功能至关重要。耐药微生物已成为并将继续成为主要的公共卫生威胁。拟议的研究将产生一个明确的酶进化模型，并增强我们对耐药性如何进化的理解。