Noncanonical reactions catalyzed by atypical flavoenzymes

非典型黄素酶催化的非典型反应

基本信息

批准号：
1506206
负责人：
Pablo Sobrado
金额：
$ 48万
依托单位：
Virginia Polytechnic Institute and State University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1506206&HistoricalAwards=false
关键词：
Noncanonical reactions catalyzed atypical flavoenzymes

项目摘要

With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Molecular Biophysics Cluster in the Molecular and Cellular Biosciences Division are funding Dr. Pablo Sobrado from Virginia Tech and Dr. John J. Tanner from the University of Missouri-Columbia to study a new class of flavoenzymes in which the flavin does not play a redox role in catalysis. Enzymes that contain riboflavin (vitamin B2) are known as flavoenzymes. In general, flavoenzymes utilize the properties of the flavins to catalyze oxidation/reduction chemistry. These reactions involve movement of electrons and changes in oxidation state as one compound is converted into another. Recently, new functionalities have been demonstrated for several flavoenzymes that do not involve changes in oxidation state for the flavin or the compounds involved in the reactions. For example, the flavin can act as an acid or base instead. Such flavoenzymes are referred to as atypical. One focus of the project is to discover the protein-flavin interactions that tune the flavin for this novel reactivity. The second objective is the characterization of 2-haloacrylate hydratase (2-HAH), an atypical flavoenzyme involved in bioremediation of halogenated compounds. Preliminary data suggest that the way the 2-HAH reaction is catalyzed may be unprecedented. Graduate and undergraduate students participating in this project will gain interdisciplinary training in chemistry, biochemistry, and structural biology. The class of atypical flavoenzymes has been much less studied compared to traditional flavin oxidoreductases. The overarching goal of this proposal is to identify the conserved three-dimensional structural features of atypical flavoenzymes that enable the flavin to be a nucleophile, electrophile, or acid/base rather than a traditional redox cofactor. Two atypical flavoenzymes will be studied: 1) UDP-galactopyranose mutase (UGM) and 2) 2-haloacrylate hydratase (2-HAH). The main goal is to formulate the biochemical and structural principles that govern the chemical reactivity of atypical flavoenzymes. The analysis of the available structural and mechanistic data on UGM resulted in a hypothesis about the structural and dynamic elements that endow flavins with new chemical activities. This hypothesis will be tested using site directed mutagenesis of UGM, X-ray crystallography, and molecular dynamics simulations. 2-HAH is a recently identified flavoprotein that catalyzes the formation of pyruvate from 2-haloacrylate. There is no net-redox change in this reaction; however, there is an absolute requirement for the reduced flavin for activity. In addition, light greatly enhances the rate of reduction of the enzyme by NAD(P)H, which is not normally seen for atypical flavoenzymes. The role of the reduced flavin in the 2-HAH reaction and the mechanism of light activation will be investigated. X-ray crystallography will be employed to solve the three-dimensional structure of 2-HAH.

凭借该奖项，化学部的生命过程化学项目和分子和细胞生物科学部的分子生物物理学集群正在资助弗吉尼亚理工大学的 Pablo Sobrado 博士和密苏里大学哥伦比亚分校的 John J. Tanner 博士研究一类新的黄素酶，其中黄素在催化中不发挥氧化还原作用。含有核黄素（维生素 B2）的酶被称为黄素酶。一般来说，黄素酶利用黄素的特性来催化氧化/还原化学。当一种化合物转化为另一种化合物时，这些反应涉及电子的移动和氧化态的变化。最近，已经证明了几种黄素酶的新功能，这些功能不涉及黄素或参与反应的化合物的氧化态变化。例如，黄素可以充当酸或碱。此类黄酶被称为非典型黄酶。该项目的一个重点是发现蛋白质-黄素的相互作用，从而调节黄素的这种新颖的反应性。第二个目标是 2-卤代丙烯酸酯水合酶 (2-HAH) 的表征，这是一种参与卤代化合物生物修复的非典型黄素酶。初步数据表明2-HAH反应的催化方式可能是前所未有的。参与该项目的研究生和本科生将获得化学、生物化学和结构生物学的跨学科培训。与传统黄素氧化还原酶相比，对非典型黄素酶类别的研究要少得多。该提案的总体目标是确定非典型黄素酶的保守三维结构特征，使黄素成为亲核试剂、亲电子试剂或酸/碱，而不是传统的氧化还原辅因子。将研究两种非典型黄酶：1) UDP-吡喃半乳糖变位酶 (UGM) 和 2) 2-卤代丙烯酸酯水合酶 (2-HAH)。主要目标是制定控制非典型黄素酶化学反应性的生化和结构原理。对 UGM 现有结构和机制数据的分析得出了关于赋予黄素新化学活性的结构和动态元素的假设。这一假设将通过 UGM 定点诱变、X 射线晶体学和分子动力学模拟进行测试。 2-HAH 是最近发现的一种黄素蛋白，可催化 2-卤代丙烯酸酯形成丙酮酸。该反应中没有净氧化还原变化；然而，对于活性而言，绝对需要减少黄素。此外，光极大地提高了 NAD(P)H 还原酶的速率，这对于非典型黄素酶来说通常是看不到的。将研究还原黄素在2-HAH反应中的作用和光活化机制。 X射线晶体学将用于解析2-HAH的三维结构。