Battling AIDS via Mechanistic Understanding of the tRNA Phe modification enzyme T

通过对 tRNA Phe 修饰酶 T 的机制理解来对抗艾滋病

基本信息

批准号：
8229462
负责人：
Lei Li
金额：
$ 22.93万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8229462
关键词：
Acquired Immunodeficiency Syndrome Anabolism Animals Biochemical Biochemical Process Biological Assay Catalysis Cells Characteristics Chemicals Chemistry Cleaved cell DNA Repair Enzymes Development Disease Electrons Enzymatic Biochemistry Enzyme Kinetics Enzymes Epidemic Family Foundations Future Goals Grant Guanine HIV Investigation Ions Iron Knowledge Laboratories Learning Ligands Light Methods Modification Molecular Nature Oligonucleotides Oxygen Pathway interactions Phenylalanine Phenylalanine-Specific tRNA Plants Population Positioning Attribute Pyruvate RNA RNA-Directed DNA Polymerase Reaction Research Resources S-Adenosylmethionine Societies Structure Subfamily lentivirinae Sulfur Techniques Testing Thymine Dimers Time Transfer RNA Translations Virus Virus Replication Y base analog chemical synthesis design enzyme mechanism enzyme substrate experience fundamental research microorganism novel novel strategies oxidation planetary Atmosphere prevent repaired research study spore photoproduct lyase wybutosine

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this research is to reveal the missing substrate "X" and shed light on the reaction mechanism for a phenylalanine transfer RNA (tRNAPhe) modification enzyme TYW1. TYW1 catalyzes the N1- methylguanine (m1G) modification to imG-14, a guanine derivative with a tricyclic aromatic ring. This reaction is the key step in wybutosine (base Y) biosynthetic pathway. Malfunction of TYW1 is suggested in HIV infected cells, which leads to the absence of base Y in tRNAPhe and causes -1 frame shifting to increase 400% during translation. This frame shifting is the ONLY way for HIV to produce reverse transcriptase, the key enzyme for virus replication. Thus, elucidating the mechanism of TYW1 via fundamental research is of great significance to the understanding of AIDS development and could potentially provide a novel approach in battling this deadly disease. TYW1 is found to possess a C-X3-C-X2-C motif, which is the characteristic feature of the radical SAM superfamily. The enzymes in this family utilize a unique [4Fe-4S] cluster to reductively cleave the S- adenosylmethionine, generating the 52-deoxyadenosyl (52-dA) radical. However, how this 52-dA radical catalyzes the N1-methylguanine modification in TYW1 is unclear due to the unknown nature of the second enzyme substrate "X". This proposal is therefore devoted to revealing the substrate "X" and removing the last obstacle in mechanistic elucidation of this important enzyme. Chemical, biochemical, spectroscopic, and enzyme kinetic methods will be employed in our experimental approach. The TYW1 enzyme expressed from different resources will be utilized in this study. In addition, tRNAPhe containing either m1G or m1G analog prepared via chemical synthesis will be employed in our investigation as well. These experiments will enable us to uncover the nature of substrate "X". The revealed structure of "X" could additionally shed light on the reaction mechanism of TYW1, which will be tested in this proposal as well as in the future investigations after the R21 grant period. PUBLIC HEALTH RELEVANCE: Malfunction of enzyme TYW1 results in the absence of base Y, a guanine derivative, at the position 37 of phenylalanine transfer RNA, which subsequently enables the HIV to produce reverse transcriptase, the key enzyme for virus replication. The mechanistic elucidation of TYW1 is hindered by an unknown substrate "X" and this project is devoted to revealing its nature and removing the major obstacle in studying TYW1 catalysis. A better understanding of TYW1 may eventually enable its induction and resume the Y biosynthesis in the HIV infected cells, providing a novel approach to battle AIDS.

描述（由申请人提供）：本研究的总体目标是揭示缺失的底物“X”并阐明苯丙氨酸转移RNA（tRNAPhe）修饰酶TYW1的反应机制。 TYW1 催化 N1-甲基鸟嘌呤 (m1G) 修饰为 imG-14，imG-14 是一种具有三环芳环的鸟嘌呤衍生物。该反应是威布托辛（Y基）生物合成途径的关键步骤。 HIV感染的细胞中发现TYW1出现故障，导致tRNAPhe中Y碱基缺失，并导致翻译过程中-1移码增加400%。这种移码是 HIV 产生逆转录酶（病毒复制的关键酶）的唯一途径。因此，通过基础研究阐明TYW1的机制对于了解艾滋病的发展具有重要意义，并可能为对抗这种致命疾病提供新的方法。 TYW1被发现具有C-X3-C-X2-C基序，这是激进SAM超家族的特征。该家族中的酶利用独特的 [4Fe-4S] 簇还原性裂解 S-腺苷甲硫氨酸，产生 52-脱氧腺苷 (52-dA) 自由基。然而，由于第二种酶底物“X”的性质未知，该52-dA自由基如何催化TYW1中的N1-甲基鸟嘌呤修饰尚不清楚。因此，该提案致力于揭示底物“X”并消除这种重要酶的机械阐明中的最后一个障碍。我们的实验方法将采用化学、生物化学、光谱和酶动力学方法。本研究将利用从不同来源表达的 TYW1 酶。此外，含有 m1G 或通过化学合成制备的 m1G 类似物的 tRNAPhe 也将用于我们的研究中。这些实验将使我们能够揭示底物“X”的性质。 “X”的揭示结构还可以进一步阐明 TYW1 的反应机制，该机制将在本提案以及 R21 资助期后的未来研究中进行测试。公共健康相关性：TYW1 酶功能障碍导致苯丙氨酸转移 RNA 37 位碱基 Y（一种鸟嘌呤衍生物）缺失，随后使得 HIV 能够产生逆转录酶（病毒复制的关键酶）。 TYW1的机制阐明受到未知底物“X”的阻碍，该项目致力于揭示其本质并消除研究TYW1催化的主要障碍。更好地了解 TYW1 最终可能能够在 HIV 感染的细胞中诱导并恢复 Y 生物合成，从而提供一种对抗艾滋病的新方法。