Structure and Mechanism in the Tautomerase Superfamily

互变酶超家族的结构和机制

基本信息

批准号：
8117692
负责人：
CHRISTIAN P. WHITMAN
金额：
$ 25.18万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8117692
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 Health 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 Play Prevalence Principal Investigator Process Proline Public Health Resistance Resources Role Route Staging Structure Structure-Activity Relationship System 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

项目摘要

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）：互变异酶超家族，一组结构上同源的蛋白质，它们具有共同的构件（2-1-2序列）（2-1-2序列）和催化氨基末端脯氨酸（Pro-1），丰富了机械，结构性和进化性问题。该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。在最后一个资金期间，为三个超家族成员建立了机制和结构[Isomer特异性的3-氯丙烯酸去核酸酶，指定为CAAD和CIS-CAAD，以及Malonate半甲醛脱羧酶（MSAD）]，显示了自然如何创建2-2-2型构图来创建结构和机械的多样性。现在，该阶段将使用这些酶作为实验工具来解决有关酶如何工作，如何发展以及新活动的出现的基本问题。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基序创建新的活动和结构。我们的主要具体目的是（1）在三个活跃位点建立底物方向和相互作用；（2）描述CAAD，顺式-CAAD和MSAD中关键突变的后果；（3）对CAAD和CIS-CAAD进行稳态的状态动力学分析；（4）检查催化杂交在互变异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模板生成酶活性的阶段。这些研究将增强我们对酶机制和细菌代谢的理解，从而更好地理解催化杂交在发散进化中所起的作用，并有助于设计基于环保的基于脯氨酸的生物催化剂。了解酶如何由于抗生素耐药细菌和其他耐药生物（例如结核和艾滋病毒）的流行而进化至关重要。一种耐药机制涉及药物的酶活性（例如2-腹坦水解）。耐药酶可以通过在生理酶中的低级耐药活性扩增来发展。因此，一个定义明确的发散进化模型是了解抗药性活动如何发展的宝贵资源，并可以提出更有效的策略来克服耐药的生物。公共卫生相关性：重要的是要了解酶如何进化和获取新功能，这是由于抗生素耐药细菌的普遍性和其他抗药性生物（如结核分枝杆菌和HIV）而言至关重要。耐药的生物已成为主要的公共卫生威胁，并将继续成为一种。拟议的研究将导致一个定义明确的酶进化模型，并增强我们对耐药性如何发展的理解。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A pre-steady state kinetic analysis of the αY60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.

DOI：
10.1021/bi3010686
发表时间：
2012-11-20
期刊：
Biochemistry
影响因子：
2.9
作者：
Huddleston JP;Schroeder GK;Johnson KA;Whitman CP
通讯作者：
Whitman CP

Kinetic, mutational, and structural analysis of malonate semialdehyde decarboxylase from Coryneform bacterium strain FG41: mechanistic implications for the decarboxylase and hydratase activities.

DOI：
10.1021/bi400567a
发表时间：
2013-07-16
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Guo, Youzhong;Serrano, Hector;Poelarends, Gerrit J.;Johnson, William H., Jr.;Hackert, Marvin L.;Whitman, Christian P.
通讯作者：
Whitman, Christian P.

A mutational analysis of the active site loop residues in cis-3-Chloroacrylic acid dehalogenase.

DOI：
10.1021/bi4004414
发表时间：
2013-06-18
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Schroeder, Gottfried K.;Huddleston, Jamison P.;Johnson, William H., Jr.;Whitman, Christian P.
通讯作者：
Whitman, Christian P.

Reaction mechanism of cis-3-chloroacrylic acid dehalogenase: a theoretical study.

DOI：
10.1021/bi900879a
发表时间：
2009-10-13
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Sevastik, Robin;Whitman, Christian P.;Himo, Fahmi
通讯作者：
Himo, Fahmi

Reaction of cis-3-chloroacrylic acid dehalogenase with an allene substrate, 2,3-butadienoate: hydration via an enamine.

DOI：
10.1021/ja206873f
发表时间：
2012-01-11
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Schroeder, Gottfried K.;Johnson, William H., Jr.;Huddleston, Jamison P.;Serrano, Hector;Johnson, Kenneth A.;Whitman, Christian P.
通讯作者：
Whitman, Christian P.