Structure, Function and Inhibition of [4Fe-4S] Proteins

[4Fe-4S]蛋白的结构、功能和抑制

基本信息

批准号：
8758404
负责人：
Eric Oldfield
金额：
$ 35.69万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8758404
关键词：
Acids Aconitate Hydratase Address Amino Acids Anabolism Anaerobic Bacteria Bacteria Binding Binding Proteins Bioinformatics Botulism Branched-Chain Amino Acids Calorimetry Catalysis Cells Chimera organism Computer Simulation Crystallography Data Dehydration Dicarboxylic Acids Dihydroxyacetone Phosphate Diphosphates Drug Targeting Electron Nuclear Double Resonance Electron Transport Enzymes Ferredoxin Freezing Gastric ulcer Grant Growth Helicobacter pylori Herbicides Human Human Microbiome Hydro-Lyases Hydroxylation Investigation Iron Isomerase Knock-out Lead Leucine Ligands Malaria Messenger RNA Metalloproteins Methionine Mycobacterium tuberculosis Organism Oxidoreductase Oxygen Parasites Pathway interactions Pharmaceutical Preparations Plants Proteins Quinolinic Acid Reaction Reporting Research Ribosomes Role Sampling Series Site-Directed Mutagenesis Spectrum Analysis Stomach Carcinoma Structure Testing Tetanus Translation Initiation Tuberculosis Tuberculosis Vaccines Water Work X-Ray Crystallography aminoacid biosynthesis base chemical reaction dehydroxylation design electron tomography fumarase A inhibitor/antagonist interest isopentenyl pyrophosphate isoprenoid isopropylmalate isomerase macrophage mutant novel novel therapeutics particle pathogen polypeptide public health relevance pyridine quinolinate ribosomal protein S1 sensor vaccine development

项目摘要

DESCRIPTION (provided by applicant): The objective of this work is to investigate the structure, mechanism of action, and inhibition of three different types of metalloprotein, all of which utilize [4Fe-4S] clusters in catalysis. The reactions catalyzed are reductive dehydroxylations, dehydrations and isomerizations, and all of the proteins are of long-term interest because they are essential for the survival of many pathogens and are not used by humans. The proteins are IspG and IspH, involved in reductive dehydroxylations in isoprenoid biosynthesis; quinolinate synthase (NadA), involved in NAD biosynthesis; and dihydroxyacid dehydratase (DHAD) and isopropylmalate isomerase (LeuCD), involved in amino-acid biosynthesis. In Aim 1 the first objective is to investigate a previously uncharacterized class of IspHs that are found in many anaerobic bacteria, such as those which cause tetanus and botulism, as well as many which are found in the human microbiome. These proteins are 2-3x larger than most bacterial IspHs and contain an IspH-RPS1 (ribosomal binding protein S1) fusion and might act as iron or oxygen sensors. The second objective is to probe the mechanisms of action of both IspG and IspH by using rapid freeze-quench EPR, calorimetry, site-directed mutagenesis, M¿ssbauer, X-ray crystallography, and DFT to characterize reaction intermediates. The third objective is to determine the structures of the 3-domain IspGs and develop novel IspG inhibitors. Aim 2 involves investigation of NadA, an enzyme whose apo-structure resembles that of IspH but whose structure with its 4Fe-4S cluster is unknown. The objective is to determine its structure and mechanism of action and to develop inhibitors as drug leads (against the organism that causes gastric ulcers and carcinoma). The third Aim involves two [4Fe-4S] hydratase/isomerases (DHAD and LeuCD). Both are predicted to contain two domains and it appears that there are structural similarities between DHAD, LeuCD, aconitase and fumarase A, a hypothesis we will test. Inhibitors have been reported as herbicides, blocking leucine and other branched chain amino-acid biosynthesis, an effect that with the LeuCD inhibitor is reversed with L- leucine, and M. tuberculosis leuCD knockouts are being developed as TB vaccines, making LeuCD and DHAD potential new drug targets and here, we will develop inhibitors, active in cells, that bind to their [4Fe-4S] clusters.

描述（由申请人提供）：这项工作的目的是研究三种不同类型金属蛋白的结构、作用机制和抑制作用，所有这些金属蛋白都利用[4Fe-4S]簇进行催化，催化的反应是还原性脱羟基。、脱水和异构化，所有这些蛋白质都具有长期意义，因为它们对于许多病原体的生存至关重要，并且不被人类利用。 IspH，参与类异戊二烯生物合成的还原脱羟基；喹啉酸合酶（NadA），参与 NAD 生物合成；以及二羟酸脱水酶（DHAD）和异丙基苹果酸异构酶（LeuCD），参与氨基酸生物合成。一类以前未表征过的 IspHs，存在于许多厌氧细菌，例如引起破伤风和肉毒杆菌中毒的细菌，以及在人类微生物组中发现的许多细菌。这些蛋白质比大多数细菌 IspH 大 2-3 倍，并且含有 IspH-RPS1（核糖体结合蛋白 S1）融合体，并且可能。作为铁或氧传感器，第二个目标是通过使用快速冷冻淬灭 EPR、量热法来探究 IspG 和 IspH 的作用机制。定点诱变，M¿第三个目标是确定 3 结构域 IspG 的结构并开发新型 IspG 抑制剂，目标 2 涉及 NadA（一种脱辅基结构类似于 NadA 的酶）。 IspH，但其 4Fe-4S 簇的结构未知，目的是确定其结构和作用机制，并开发作为药物先导物的抑制剂（针对引起的生物体）。第三个目标涉及两种 [4Fe-4S] 水合酶/异构酶（DHAD 和 LeuCD），预计 DHAD、LeuCD、乌头酸酶和延胡索酸酶 A 之间存在结构相似性。据报道，抑制剂可作为除草剂，阻断亮氨酸和其他支链氨基酸的生物合成，这是 LeuCD 的作用。 L-亮氨酸逆转抑制剂，结核分枝杆菌 leuCD 敲除正在开发为结核疫苗，使 LeuCD 和 DHAD 成为潜在的新药物靶点，在这里，我们将开发在细胞中活跃的抑制剂，与它们的 [4Fe-4S] 结合集群。