Structure and Mechanism in the Tautomerase Superfamily

描述（由申请人提供）：互变酶超家族是一组结构上同源的蛋白质，具有共同的构建块（2-1-2基序）和催化氨基末端脯氨酸（Pro-1），具有丰富的机制，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，我们建立了三个超家族成员的机制和结构[同分异构体特异性3-氯丙烯酸脱盐酶，指定为CaaD和顺式CaaD，以及丙二酸半醛脱羧酶（MSAD）]，展示了Nature如何使用2-1-2基序创造结构和机制多样性。现在，利用这些酶作为实验工具来解决酶如何工作、如何进化以及新活动如何产生等基本问题的阶段已经准备好了。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主题创造新的活动和结构。我们的主要具体目标将是：(1)确定底物取向和三个活性位点的相互作用；(2)描述CaaD、顺式CaaD和MSAD关键突变的后果；(3)进行CaaD和顺式CaaD的预稳态动力学分析；(4)通过系统发育和生物信息学分析，研究催化乱交在互变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。这些研究将增强我们对酶机制和细菌代谢的理解，更好地理解催化乱交在分化进化中的作用，并有助于设计环保型脯氨酸生物催化剂。了解酶如何随着耐抗生素细菌和其他耐药生物（如结核分枝杆菌和艾滋病毒）的流行而进化是至关重要的。耐药性的一种机制涉及药物的酶失活（例如，2-内酰胺水解）。抗性酶可以通过生理酶中低水平抗性活性的扩增而进化。因此，一个定义良好的分化进化模型是了解耐药性活动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。公共卫生相关性：由于耐抗生素细菌和其他耐药生物（如结核分枝杆菌和艾滋病毒）的流行，了解酶如何进化并获得新功能至关重要。耐药生物已经成为并将继续成为一个重大的公共卫生威胁。拟议的研究将产生一个定义良好的酶进化模型，并增强我们对耐药性如何进化的理解。

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.