Exploring the Mechanism of HMG-CoA Reductase With Time-Resolved Laue Diffraction

本项目的目标是对mevalonii假单胞菌3-羟基-3-甲基戊二酰辅酶a （HMG-CoA）还原酶的分子机制进行多方面的研究。这种酶催化HMG-CoA和甲羟戊酸的相互转化，这一反应是合成活性类异戊二烯的根本途径，包括类固醇激素、焦磷酸法尼酯和胆固醇等生物学上重要的分子，以及信号转导网络的组成部分。利用该酶家族的两种类型成员，人类（I类）和P. mevalonii （II类）HMG-CoA还原酶在二元和非生产性三元配合物中的结构，已经提出了一种催化机制，现在可以在分子细节上进行研究。随着这种酶的慢底物的发现，人们可以生产出晶体复合物，从而提供一系列沿着反应途径捕获中间体的快照。这些配合物的初步实验表明，P. mevalonii还原酶在晶体中是活跃的，这开启了使用时间分辨劳厄衍射观察催化过程中结构变化的可能性。本项目将利用这些方法研究HMG-CoA还原酶机制的潜在结构原理。该项目将结合晶体学和动力学技术来研究这种多步骤酶反应的机制难题。更广泛的影响：研究工作将与对本科生和高中生的教育推广相协调。该项目为培养大学生提供了一个很好的机会。此外，还将大力参与与当地高中的合作研究工作。作为ISEF认可的区域科学与工程博览会的主任，pi与三位教授高级研究课程的当地高中教师密切合作。这些合作高中研究项目在高中教授科学和科学研究的基础知识，然后将学生与普渡大学的研究人员进行更深入的研究。私家侦探与这些教师密切合作，寻找合适的实验室匹配，在实验室接待高中研究人员，并陪同教师和学生获胜者参加州和国际博览会。这些活动将在未来继续进行。

The goal of this project is a multifaceted investigation of the molecular mechanism of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from the bacterium Pseudomonas mevalonii. This enzyme catalyzes the interconversion of HMG-CoA and mevalonate, a reaction at the root of the pathway leading to synthesis of active isoprenoids, including such biologically important molecules as steroid hormones, farnesyl pyrophosphate and cholesterol, as well as components of signal transduction networks. Using the structures of the two type members of this enzyme family, the human (Class I) and P. mevalonii (Class II) HMG-CoA reductases in binary and non-productive ternary complexes, a catalytic mechanism has been proposed that can now be investigated in molecular detail. With the discovery of a slow substrate for this enzyme, one can produce crystalline complexes that can provide a series of snapshots of trapped intermediates along the reaction pathway. Initial experiments with these complexes have shown that the P. mevalonii reductase is active in the crystal, which opens the possibility of observing the structural changes during catalysis using time-resolved Laue diffraction. This project will use these approaches to study the underlying structural principles of the mechanism of HMG-CoA reductase. The project will combine crystallographic and kinetic techniques to investigate the mechanistic puzzles of this multistep enzyme reaction.Broader Impacts: The research efforts will be coordinated with educational outreach to undergraduate and high school students. The project provides an outstanding opportunity for training of undergraduate students. In addition, there will be a strong involvement in a collaborative research effort with local high schools. As Director of the ISEF accredited Regional Science and Engineering Fair, the P.I. works closely with three local high school teachers who teach an advanced research class. These collaborative high school research programs teach the basics of science and scientific research at the high school, and then match students with researchers at Purdue for more advanced studies. The P.I. works closely with these teachers to look for appropriate laboratory matches, has hosted high school researchers in the laboratory and accompanies the teachers and student winners to the state and international fairs. These activities will continue in the future.