Structural and proton dynamics of pyridoxal-5’-phosphate dependent enzymes Resubmission (Diversity Supplement)

5-磷酸吡哆醛依赖性酶的结构和质子动力学重新提交（多样性补充）

• 批准号: 10359304
• 负责人: Leonard J Mueller
• 金额: $ 1.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359304
• 关键词: 3-Dimensional; Active Sites; Alanine Racemase; Amino Acids; Antibiotics; Antidiabetic Drugs; Antimalarials; Aspartate Transaminase; Catalysis; Coenzymes; Complement; Crystallography; Drug Design; Drug Modelings; Drug Targeting; Enzyme Inhibitor Drugs; Enzyme Kinetics; Enzymes; Family; Foundations; Glycine Hydroxymethyltransferase; Goals; Hydrogen; Isotopes; Joints; Kinetics; Knowledge; Lyase; Molecular Computations; Motion; NMR Spectroscopy; Neutrons; Pharmaceutical Preparations; Phenols; Property; Protein Dynamics; Proteins; Proteome; Protons; Pyridoxal Phosphate; Reaction; Relaxation; Research; Resolution; Role; Specificity; Structure; Techniques; Time; Transaminases; Tryptophan Synthase; Tyrosine; Visualization; Vitamin B6; X-Ray Crystallography; biophysical techniques; cofactor; design; enzyme mechanism; improved; insight; molecular dynamics; new therapeutic target; novel; parent project; pressure; protein structure; protonation; solid state nuclear magnetic resonance; vibration

Project Summary (NO CHANGE IN THE SCOPE OF THE PARENT PROJECT) PLP-dependent enzymes represent about 2% of the proteome, and a number of them are current or potential drug targets. There are four major families of pyridoxal-5’-phosphate (PLP)-dependent enzymes, distinguished by different three-dimensional folds: the aspartate aminotransferase or α-family (Fold I), the tryptophan synthase or β-family (Fold II), the alanine racemase family (Fold III), and the D-amino acid aminotransferase family (Fold IV). Using X-ray crystallography, a great deal has been learned about the role of both these enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding that limit drug design. The goal of the proposed project is to provide a very detailed understanding of PLP-dependent enzyme mechanisms by coordinately defining their structures and dynamics from the global to the atomic level. To accomplish this, we will employ a synergistic combination of biophysical techniques that are sensitive to different size- and time-scales. These will include joint X-ray/neutron crystallography, solid-state NMR spectroscopy, molecular dynamics and QM/MM calculations, inelastic neutron scattering, rapid kinetics techniques, and heavy enzyme kinetic isotope effects. We will focus on four structurally well-characterized PLP-dependent enzymes, aspartate aminotransferase, serine hydroxymethyltransferase, tyrosine phenol-lyase and tryptophan synthase, but for which information on protonation and dynamics is lacking. The enzymes are drug targets (aspartate aminotransferase, serine hydroxymethyltransferase) or serve as models for drug targets (tryptophan synthase, tyrosine phenol-lyase). These enzymes catalyze diverse reactions, but use the same cofactor in similar active sites. Thus, we postulate that the reaction specificity must be controlled by a combination of protein dynamics and selective protonation of reaction intermediates. Joint X-ray/neutron crystallography will be the foundation of our research, providing an atomic-level structural basis for protein dynamics and accurate visualization of hydrogen atoms in protein structures at moderate resolutions. The results of X-ray/neutron crystallography will be combined with novel solid-state NMR crystallography and with inelastic neutron scattering to characterize the global and local motions of these enzymes at picosecond-to- microsecond time scales. Pressure-jump relaxation kinetics, and heavy enzyme kinetic isotope effects will complement and provide dynamic information on domain motion in the picosecond to minute time-scales. It should be noted that the inelastic neutron scattering, pressure-jump and heavy enzyme kinetics are complementary techniques in that they all are sensitive to changes in the vibrational motions of the enzyme, but interrogate at different time scales. All these results will be integrated with molecular computations to provide an unprecedented complete picture of the dynamic properties of these important enzymes. This knowledge may allow the design of novel, potent and selective enzyme inhibitors that may provide new drugs targeted against PLP-dependent enzymes.

项目概要 （母项目的范围没有变化） PLP 依赖性酶约占蛋白质组的 2%，其中许多是当前或潜在的 药物靶点。 5'-磷酸吡哆醛 (PLP) 依赖性酶有四个主要家族，分别是 由不同的三维折叠：天冬氨酸转氨酶或 α-家族（折叠 I）、色氨酸 合酶或 β-家族（折叠 II）、丙氨酸消旋酶家族（折叠 III）和 D-氨基酸转氨酶 家庭（折叠 IV）。使用 X 射线晶体学，人们对这两种物质的作用有了很多了解。 催化作用中的酶和辅助因子。尽管如此，我们对限制药物的理解仍然存在严重差距 设计。拟议项目的目标是提供对 PLP 依赖的非常详细的理解 酶机制通过从整体到原子水平协调定义其结构和动力学。 为了实现这一目标，我们将采用对以下因素敏感的生物物理技术的协同组合： 不同的规模和时间尺度。这些将包括联合 X 射线/中子晶体学、固态核磁共振 光谱学、分子动力学和 QM/MM 计算、非弹性中子散射、快速动力学 技术和重酶动力学同位素效应。我们将重点关注四个结构良好的 PLP 依赖性酶、天冬氨酸氨基转移酶、丝氨酸羟甲基转移酶、酪氨酸苯酚裂解酶 和色氨酸合酶，但缺乏质子化和动力学信息。这些酶是 药物靶点（天冬氨酸转氨酶、丝氨酸羟甲基转移酶）或作为药物模型 靶标（色氨酸合酶、酪氨酸酚裂解酶）。这些酶催化不同的反应，但使用 相似的活性位点具有相同的辅因子。因此，我们假设反应特异性必须由 蛋白质动力学和反应中间体选择性质子化的结合。联合X射线/中子 晶体学将成为我们研究的基础，为蛋白质提供原子级结构基础 以中等分辨率对蛋白质结构中的氢原子进行动力学和精确可视化。这 X 射线/中子晶体学的结果将与新型固态 NMR 晶体学相结合 非弹性中子散射来表征这些酶在皮秒到皮秒的全局和局部运动 微秒时间尺度。压力跳跃弛豫动力学和重酶动力学同位素效应将 补充并提供皮秒到分钟时间尺度内域运动的动态信息。它 应该指出的是，非弹性中子散射、压力跳跃和重酶动力学是 互补技术，因为它们都对酶振动运动的变化敏感， 但在不同的时间尺度进行询问。所有这些结果将与分子计算相结合 提供了这些重要酶的动态特性的前所未有的完整图像。这 知识可以允许设计新颖的、有效的和选择性的酶抑制剂，从而提供新的药物 针对 PLP 依赖性酶。