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：
10119760
负责人：
Andrii Y Kovalevskyi
金额：
$ 66.14万
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/10119760
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 Coenzymes Complement Crystallization 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 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 Side 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 cofactor design enzyme mechanism improved insight ionization molecular dynamics molecular mechanics new therapeutic target novel oxidation pressure prospective protein structure protonation racemization 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) 的酶参与多种氨基反应酸和胺，包括转氨基、外消旋、脱羧、β-和γ-消除、β-和γ- 取代，以及最近发现的许多重要的电流或氧化作用。潜在的药物靶标是 PLP 依赖性酶，包括 γ-氨基丁酸转氨酶、多巴然而，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。目前许多针对 PLP 依赖性酶的药物由于缺乏特异性而存在副作用因此，了解这些酶与分子和原子的反应非常重要。详细程度有助于使用 X 射线晶体学设计更有效、更具选择性的新药物，尽管如此，人们对酶和辅因子在催化中的作用已经有了很多了解。我们对 PLP 依赖性酶的理解中存在的关键差距仅限制了药物设计。缺少两个重要信息：首先，它们缺少有关反应动力学的重要信息。众所周知，配体结合和催化中的蛋白质运动在酶中发挥着核心作用，但这种情况是如何发生的尚不清楚此外，在PLP催化中发挥关键作用的氢原子也不是直接未知的。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空白。一般酶的催化作用，特别是 PLP 依赖性酶的催化作用，其中活性位点质子化状态最近的天冬氨酸中子衍射结构似乎在控制反应特异性中发挥着关键作用。转氨酶在活性位点的一个意想不到的位置发现了一个质子，形成了一个低势垒氢底物羧酸盐和醛亚胺氮之间的键在我们对质子化的理解中是空白的。和电离态阻碍了治疗剂的合理设计，例如针对特定情况定制的治疗剂拟议项目的目标是提供对 PLP 的非常详细的了解。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：不同的尺寸和时间尺度，其中包括联合 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) 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。这一合作项目的成果将首次提供催化的全球图景。大型且核心重要的一类酶，具有真正的原子分辨率，可用于稳定的中间体以及它们之间的动态联系将是我们的结果和开发的技术的见解。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。