Noncanonical reactions catalyzed by atypical flavoenzymes

通过这一奖项，化学部的生命过程化学计划和分子和细胞生物科学部的分子生物物理组将资助弗吉尼亚理工大学的Pablo Sobrado博士和密苏里-哥伦比亚大学的John J.Tanner博士研究一种黄素在催化中不起氧化还原作用的新型黄素酶。含有核黄素(维生素B2)的酶称为黄素酶。总体而言，黄素酶利用黄素的性质来催化氧化/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运动和氧化态的变化。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状态的变化。例如，黄素可以作为酸或碱。这种黄素酶被称为非典型酶。该项目的一个重点是发现蛋白质与黄素的相互作用，从而调节黄素的这种新的反应活性。第二个目标是2-卤代丙烯酸酯水合酶(2-HAH)的特性，它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方面的跨学科培训。与传统的黄素氧化还原酶相比，对非典型黄素酶的研究要少得多。这项建议的首要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、亲电或酸/碱，而不是传统的氧化还原辅因子。将研究两种非典型黄素酶：1)UDP-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的生化和结构原理。对UGM的现有结构和机理数据的分析得出了一个假说，即赋予黄素新的化学活性的结构和动态元件。这一假说将通过UGM的定点突变、X射线结晶学和分子动力学模拟来验证。2-HAH是新近发现的一种黄素蛋白，可催化2-卤代丙烯酸酯合成丙酮酸。在这个反应中没有净氧化还原的变化；然而，活性绝对需要还原的黄素。此外，光大大提高了NAD(P)H对酶的还原速度，这在非典型黄素酶中是不常见的。研究了还原黄素在2-HAH反应中的作用和光活化机理。2-HAH的三维结构将采用X射线结晶学的方法进行解析。

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.