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.

项目摘要 （父母项目的范围没有更改） PLP依赖性酶约占蛋白质组的2％，其中许多是当前或电势 药物靶标。有四个主要的吡啶还原-5'-磷酸（PLP）依赖性酶，有特色的酶 通过不同的三维褶皱：天冬氨酸氨基转移酶或α-家庭（折叠I），色氨酸 合酶或β-家庭（FOLD II），丙氨酸种族酶家族（折叠III）和D-氨基酸氨基转移酶 家庭（折叠IV）。使用X射线晶体学，已经了解了这两个的作用 催化中的酶和辅因子。尽管如此，我们的理解中仍然存在关键的差距 设计。拟议项目的目的是提供对PLP依赖性的非常详细的理解 酶机制通过协调定义从全球到原子水平的结构和动态。 为此，我们将采用对生物物理技术敏感的生物物理技术的协同组合 不同的尺寸和时间尺度。这些将包括关节X射线/中子晶体学，固态NMR 光谱，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。我们将重点放在四个结构上良好的 PLP依赖性酶，天冬氨酸氨基转移酶，丝氨酸羟基转移酶，酪氨酸酚 - 洛碱基 和色氨酸合酶，但缺乏有关质子化和动力学的信息。酶是 药物靶标（天冬氨酸氨基转移酶，丝氨酸羟基转移酶）或作为药物的模型 靶标（色氨酸合酶，酪氨酸酚 - 叶酶）。这些酶会催化潜水反应，但使用 在相似的活性位点相同的辅助因子。那就是我们假设反应特异性必须由 蛋白质动力学和反应中间体的选择性质子化的组合。关节X射线/中子 晶体学将是我们研究的基础，为蛋白质提供了原子水平的结构基础 中等分辨率蛋白质结构中氢原子的动力学和准确可视化。这 X射线/中子晶体学的结果将与新型的固态NMR晶体学结合，并与 非弹性中子散射以表征这些酶在皮秒到第一的全球和局部运动 微秒时间尺度。压力悬浮的放松动力学和重酶动力学效应将 补充并提供有关域运动至分钟尺度中域运动的动态信息。 应注意的是，非弹性中子散射，压力跳跃和重型酶动力学是 完全的技术是，它们都对酶的振动运动的变化敏感， 但是在不同的时间尺度上审问。所有这些结果将与分子计算集成到 提供这些重要酶的动态特性的前所未有的完整图片。 知识可能允许设计新型，潜在和选择性酶抑制剂，这些酶抑制剂可能提供新药 针对依赖PLP的酶的靶向。