Structure and Mechanism in the Tautomerase Superfamily

描述(申请人提供)：互变擦除酶超家族，一组结构上同源的蛋白质，共享一个共同的构建块(2-1-2基序)和一个催化氨基末端脯氨酸(Pro-1)，包含丰富的机械性、结构性和进化性问题。该项目的长期目标是通过结合机械酶学、分子生物学、X射线结晶学和生物信息学来解决这些问题。在上一次供资期间，建立了三个超家族成员的机制和结构[特定异构体的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)]，表明大自然如何利用2-1-2基序来创造结构和机制的多样性。现在，我们将使用这些酶作为实验工具，解决有关酶如何工作、如何进化以及如何产生新活动的基本问题。拟议的研究将确定在这个系统中使用的基本原则，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。我们的主要目标是：(1)确定三个活性部位的底物定向和相互作用；(2)描述CAAD、cis-CAAD和MSAD关键突变的后果；(3)进行CAAD和cis-CAAD的稳态前动力学分析；以及(4)通过系统发育和生物信息学分析，研究催化混杂在互变擦除酶超家族进化中的作用，并建立进化关系。该结果为使用2-1-2模板产生酶活性奠定了基础。这些研究将加深我们对酶机制和细菌代谢的了解，使我们更好地理解催化杂乱在分化进化中所起的作用，并有助于设计环境友好的基于脯氨酸的生物催化剂。了解由于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)的流行而导致的酶如何进化是至关重要的。耐药性的一种机制涉及药物的酶失活(例如，2-内酰胺水解法)。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，一个明确的分化进化模型是了解耐药活动最初如何进化的宝贵资源，并可能为克服耐药生物提出更有效的策略。与公共卫生相关：由于抗生素耐药细菌和其他耐药生物如结核分枝杆菌和艾滋病毒的流行，了解酶是如何进化和获得新功能的至关重要。耐药生物已经成为并将继续成为一个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，并首先加强我们对耐药性进化方式的理解。

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.