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

5-磷酸吡哆醛依赖性酶的结构和质子动力学重新提交（设备补充）

• 批准号: 10387748
• 负责人: Leonard J Mueller
• 金额: $ 5.33万
• 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/10387748
• 关键词: 4-Aminobutyrate aminotransferase; Acids; Active Sites; Alanine Racemase; Amines; Amino Acids; Antibiotics; Antidiabetic Drugs; Antimalarials; Aspartate Transaminase; Catalysis; Coenzymes; Crystallization; Crystallography; DOPA decarboxylase; Decarboxylation; Drug Design; Drug Targeting; Electrostatics; Environment; Enzymes; Equipment; Glycine Hydroxymethyltransferase; Goals; Hydrogen; Hydrogen Bonding; Joints; Kinetics; Ligand Binding; Molecular; Motion; Neutron Diffraction; Neutrons; Nitrogen; Ornithine Decarboxylase; Pharmaceutical Preparations; Play; Positioning Attribute; Proteins; Protons; Pyridoxal Phosphate; Quantum Mechanics; Reaction; Resolution; Role; Specificity; Structure; Techniques; Therapeutic Agents; Time; Vitamin B6; X-Ray Crystallography; base; biophysical techniques; carboxylate; design; enzyme mechanism; improved; insight; ionization; molecular dynamics; molecular mechanics; novel; oxidation; prospective; protonation; racemization; side effect; solid state nuclear magnetic resonance; transamination

Enzymes containing pyridoxal-5'-phosphate (PLP) are involved in a broad range of reactions of amino acids and amines, including transamination, racemization, decarboxylation, β- and γ-elimination, β- and γ- substitution, and, as recently discovered, even oxidation and oxygenation. A number of important current or prospective drug targets are PLP-dependent enzymes, including γ-aminobutyrate aminotransferase, DOPA decarboxylase, alanine racemase, ornithine decarboxylase, and serine hydroxymethyltransferase. However, many of the current drugs that target PLP-dependent enzymes suffer from side effects due to lack of specificity for their targets. Thus, it is important to understand the reactions of these enzymes with molecular and atomic levels of detail to help in the design of new more potent and more selective drugs. Using X-ray crystallography, a great deal has been learned about the role of both enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding of PLP-dependent enzymes that limit drug design. Crystal structures alone are missing two essential pieces of information. First, they lack important information regarding reaction dynamics. Protein motion in ligand binding and catalysis is known to play a central role in enzymes, but how this occurs is essentially unknown. In addition, hydrogen atoms that play critical roles in PLP catalysis are not directly observed by X-ray crystallography. This leaves a significant gap in our understanding of general acid-base catalysis in enzymes in general and particularly in PLP-dependent enzymes, where active site protonation states appear to play critical roles in control of reaction specificity. A recent neutron diffraction structure of aspartate aminotransferase found a proton in an unpredicted position in the active site, forming a low barrier hydrogen bond between the substrate carboxylate and the aldimine nitrogen. This void in our understanding of protonation and ionization states impedes rational design of therapeutic agents that, for example, are tailored for specific electrostatic environments. The goal of the proposed project is to provide a very detailed understanding of PLP 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 crystallography, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations, inelastic neutron scattering, steady-state and rapid kinetics techniques of PLP dependent enzymes. The results of this collaborative venture will provide, for the very first time, a global picture of catalysis by a large and centrally important class of enzymes at true atomic-resolution for stable intermediates as well as the dynamic connections between them. The insights from our results and the techniques developed will be transferable to many other enzymes, and may contribute to improved rational drug design of novel antibiotic, antidiabetic, antimalarial, and other drugs.

含有吡ido-5'-磷酸（PLP）的酶参与了广泛的氨基反应 酸和胺，包括转移，种族化，脱羧，β-和γ-脱羧化，β-和γ- 替代，并且正如最近发现的，甚至是氧化物和氧合。许多重要电流或 前瞻性药物靶标是PLP依赖性酶，包括γ-氨基丁酸氨基酸氨基转移酶DOPA 脱羧酶，丙氨酸种族酶，鸟氨酸脱羧酶和丝氨酸羟基转移酶。然而， 由于缺乏特异性，靶向PLP依赖性酶的许多当前药物都遭受副作用 对于他们的目标。这是重要的是要了解这些酶与分子和原子的反应 细节的水平，以帮助设计新的潜力和更多选择性药物。使用X射线晶体学， 关于酶和辅因子在催化中的作用，已经了解了很多。尽管如此，仍然有 我们对限制药物设计的PLP依赖性酶的理解的关键差距。仅晶体结构是 缺少两个基本信息。首先，他们缺乏有关反应动态的重要信息。 已知配体结合和催化中的蛋白质运动在酶中起着核心作用，但是这种情况的发生是 本质上是未知的。此外，在PLP催化中起关键作用的氢原子不是直接的 通过X射线晶体学观察。这在我们对一般酸碱的理解中留下了很大的差距 一般，尤其是PLP依赖性酶中的酶催化，活性位点质子化状态 似乎在控制反应特异性方面起着关键作用。天冬氨酸的最新中子衍射结构 氨基转移酶在活性位点发现了一个无法预测的位置的质子，形成了低屏障氢 基材羧酸盐和醛氮氮之间的键。这在我们对质子化的理解中的空白 电离状态阻碍了治疗剂的合理设计，例如，该设计是针对特定的 静电环境。拟议项目的目的是提供对PLP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 晶体学，分子动力学（MD）和量子力学/分子力学（QM/mm） PLP依赖性酶的计算，非弹性中子散射，稳态和快速动力学技术。 这项合作企业的结果将首次提供全球催化的图片 在稳定中间体的真原子分辨率和 它们之间的动态连接。我们的结果和开发技术的见解将是 可转移到许多其他酶，并可能有助于改善新型抗生素的合理药物设计 抗糖尿病，抗疟疾和其他药物。