Structure and Mechanism in the Tautomerase Superfamily

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，具有共同的结构单元（2-1-2基序）和催化性氨基末端脯氨酸（Pro-1），富含机制、结构和进化问题。这个项目的长期目标是通过机械酶学、分子生物学、X射线晶体学和生物信息学的结合来解决这些问题。在上一个资助期内，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cis-CaaD，以及丙二酸半醛脱羧酶（MSAD）]的机制和结构被建立起来，显示了自然如何使用2-1-2基序来创造结构和机制多样性。现在的阶段是使用这些酶作为实验工具，以解决酶如何工作，它们如何进化以及新活动如何出现的基本问题。拟议的研究将确定该系统中使用的基本原理，以便我们最终模仿自然的过程，并使用2-1-2基序创造新的活动和结构。我们的主要目标是：（1）确定三个活性位点的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的稳态前动力学分析;以及（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不同进化中所起的作用，并有助于设计环境友好的基于脯氨酸的生物催化剂。了解酶是如何进化的是至关重要的，因为耐药细菌和其他耐药生物体（例如，M.结核病和艾滋病毒）。耐药性的一种机制涉及药物的酶失活（例如，2-内酰胺水解）。抗性酶可以通过放大生理酶中的低水平抗性活性而进化。因此，一个定义明确的趋异进化模型是了解耐药性活动如何首先演变的宝贵资源，并可以提出克服耐药生物体的更有效策略。公共卫生相关性：由于耐药细菌和其他耐药生物如M.肺结核和艾滋病。耐药微生物已成为一个主要的公共卫生威胁，并将继续成为一个威胁。拟议的研究将为酶的进化提供一个明确的模型，并增强我们对抗性如何首先进化的理解。

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.