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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：
10679219
负责人：
Andrii Y Kovalevskyi
金额：
$ 5.86万
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/10679219
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 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）的酶参与了广泛的氨基反应