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Structural and proton dynamics of pyridoxal-5'-phosphate dependent enzymes (resubmission)

5-磷酸吡哆醛依赖性酶的结构和质子动力学（重新提交）

基本信息

批准号：
10688203
负责人：
Andrii Y Kovalevskyi
金额：
$ 59.64万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
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/10688203
关键词：
3-Dimensional 3-hydroxybutanal 4-Aminobutyrate aminotransferase Acids Active Sites Affect Alanine Racemase Amines Amino Acids Antibiotics Antidiabetic Drugs Antimalarials Aspartate Transaminase Catalysis Complement Crystallography DOPA decarboxylase Decarboxylation Drug Design Drug Modelings Drug Targeting Electrostatics Environment Enzyme Inhibitor Drugs Enzyme Kinetics Enzymes Family Foundations Glycine Hydroxymethyltransferase Goals Hydrogen Hydrogen Bonding Hydroxymethyltransferases Isotopes Joints Kinetics Knowledge Learning Ligand Binding Location Lyase Molecular Molecular Computations Motion Movement NMR Spectroscopy Neutron Diffraction Neutrons Nitrogen Ornithine Decarboxylase Pharmaceutical Preparations Phenols Play Positioning Attribute Property Protein Dynamics Proteins Proteome Protons Pyridoxal Phosphate Quantum Mechanics Reaction Relaxation Research Resolution Role Serine Specificity Spectrum Analysis Structure Techniques Therapeutic Agents Time Transaminases Tryptophan Synthase Tyrosine Visualization Vitamin B6 X-Ray Crystallography base beta pleated sheet biophysical techniques carboxylate carboxylation cofactor design enzyme mechanism improved insight ionization molecular dynamics molecular mechanics new therapeutic target novel oxidation pressure prospective protein structure protonation racemization rational design side effect solid state nuclear magnetic resonance transamination vibration

项目摘要

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.

含有吡哆醛-5 '-磷酸（PLP）的酶参与氨基的广泛反应。酸和胺，包括转氨基作用、外消旋作用、脱羧作用、β-和γ-消除作用、β-和γ- 取代，以及最近发现的氧化和氧化。一些重要的当前或潜在的药物靶点是PLP依赖性酶，包括γ-氨基丁酸转氨酶、多巴脱羧酶、丙氨酸消旋酶、鸟氨酸脱羧酶和丝氨酸羟甲基转移酶。然而，在这方面，目前许多靶向PLP依赖性酶的药物由于缺乏特异性而具有副作用他们的目标。因此，了解这些酶与分子和原子的反应是很重要的。帮助设计新的更有效和更有选择性的药物。利用X射线晶体学，关于酶和辅因子在催化中的作用，人们已经了解了很多。尽管如此，我们对限制药物设计的PLP依赖性酶的理解存在重大差距。晶体结构本身就是缺少了两个重要的信息首先，它们缺乏关于反应动力学的重要信息。已知蛋白质在配体结合和催化中的运动在酶中起核心作用，但这是如何发生的，基本上未知。此外，在PLP催化中起关键作用的氢原子不直接与PLP反应。通过X射线晶体学观察。这就给我们对一般酸碱平衡的理解留下了很大的空白一般酶中的催化，特别是在PLP依赖性酶中，其中活性位点质子化状态似乎在反应特异性的控制中起关键作用。天冬氨酸的中子衍射结构氨基转移酶发现一个质子在一个意想不到的位置在活性位点，形成一个低势垒氢底物羧酸盐和醛亚胺氮之间的键。我们对质子化的理解并且电离状态阻碍了治疗剂的合理设计，例如，静电环境拟议项目的目标是提供一个非常详细的了解PLP 酶的机制，通过协调定义其结构和动力学从全球到原子水平。为了实现这一点，我们将采用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，固态核磁共振晶体学、分子动力学（MD）和量子力学/分子力学（QM/MM）计算，非弹性中子散射，稳态和快速动力学技术的PLP依赖酶。这一合作项目的结果将首次提供一个全球性的催化图景，在真正的原子分辨率的大的和中心重要的一类酶的稳定的中间体，以及它们之间的动态联系。从我们的研究结果和技术开发的见解将是可转移到许多其他酶，并可能有助于改进新型抗生素的合理药物设计，抗糖尿病、抗疟疾和其他药物。