Mechanism and Structure in an Enzyme Superfamily

酶超家族的机制和结构

• 批准号: 1309323
• 负责人: Dirk Iwata-Reuyl
• 金额: $ 38.2万
• 依托单位: Portland State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1309323&HistoricalAwards=false
• 关键词: Mechanism; Structure; Enzyme; Superfamily

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Dirk Iwata-Reuyl of Portland State University to investigate the molecular/structural basis for the divergent chemistries exhibited by enzymes belonging to the Tunneling-fold superfamily (T-fold). Members of the T-fold superfamily catalyze a remarkable diversity of chemistries, and while T-fold proteins typically exhibit very low sequence homology, their topological homology is extremely high. Specifically, this project addresses two T-fold enzymes involved in the biosynthesis of the modified tRNA nucleosides queuosine and archaeosine, the enzymes QueF and QueF-L. QueF catalyzes the reduction of a nitrile precursor to an amine in the queuosine pathway in Bacteria, the only example of a nitrile reduction known in biology. QueF-L exhibits high sequence homology to QueF by catalyzes the chemically distinct conversion of a nitrile to an amidino group in the archaeosine pathway in Archaea. These systems will be studied via the application of kinetic (steady state and pre-steady state), mechanistic, and X-ray structural studies of the wild-type and mutant enzymes to elucidate the chemical mechanisms, as well as the structural basis for substrate/cofactor binding and catalysis.The large number of protein structures elucidated through X-ray crystallography over the past several decades whose coordinates are available through the Protein Data Bank has revealed that the number of basic protein scaffolds responsible for the myriad proteins in biology is surprisingly small. Elucidating the molecular basis for the different functions exhibited by members in a given structural family is one of the next challenges facing structural and mechanistic biology, impacting our ability to correctly identify the function of new proteins identified by whole genome sequencing, to engineer proteins for new functions, as well as our understanding of how proteins evolve. This project focuses on elucidating the mechanistic chemistry of several members of the so-called Tunneling-fold family and identifying the protein structural elements responsible for that chemistry. The work is multidisciplinary, approaching the problem with tools from chemistry, biochemistry, and structural and molecular biology to provide an understanding of the molecular basis for the biological function of this functionally diverse family of proteins. In addition to supporting the work of graduate and undergraduate students, the project also incorporates the efforts of high school teachers through a summer outreach program that engages K12 teachers in research.

化学系的生命过程化学项目将资助波特兰州立大学的Dirk Iwata-Ruyl博士研究隧道折叠超家族(T-折叠酶)所表现出的不同化学结构的分子/结构基础。T-折叠超家族的成员催化着各种各样的化学变化，虽然T-折叠蛋白通常表现出非常低的序列同源性，但它们的拓扑同源性却非常高。具体地说，该项目涉及两种T-折叠酶，即酶QueF和QueF-L，它们参与了修饰的tRNA核苷Queuosine和Arcosine的生物合成。在细菌中，QUF催化在队列蛋白途径中将氰基前体还原为胺，这是生物学上已知的唯一的氰基还原的例子。QUEF-L通过催化古生物中古丝氨酸途径中腈到胺基的不同化学转化而显示出与QUF的高度同源性。这些体系将通过应用动力学(稳定状态和稳定前状态)、机制和X射线结构研究野生型和突变酶来阐明其化学机制，以及底物/辅因子结合和催化的结构基础。过去几十年来，通过X射线结晶学阐明的大量蛋白质结构，其坐标可以通过蛋白质数据库获得，这表明，在生物学中负责无数蛋白质的基本蛋白质支架的数量令人惊讶地少。阐明特定结构家族成员不同功能的分子基础是结构和机械生物学面临的下一个挑战之一，影响我们正确识别通过全基因组测序确定的新蛋白质的功能的能力，为新功能设计蛋白质，以及我们对蛋白质如何进化的理解。这个项目的重点是阐明所谓的隧道折叠家族的几个成员的机械化学，并确定负责该化学的蛋白质结构元件。这项工作是多学科的，利用化学、生物化学以及结构和分子生物学的工具来解决这个问题，以提供对这个功能多样化的蛋白质家族的生物功能的分子基础的理解。除了支持研究生和本科生的工作外，该项目还通过一个暑期推广计划，让K12教师参与研究，纳入了高中教师的努力。