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

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TYW 1的反应机制。TYW 1催化N1-甲基鸟嘌呤（m1 G）修饰为imG-14，一种具有三环芳环的鸟嘌呤衍生物。该反应是间布腺苷（碱基Y）生物合成途径中的关键步骤。在HIV感染的细胞中提示TYW 1的功能障碍，这导致tRNAPhe中碱基Y的缺失，并导致翻译期间-1移码增加400%。这种移码是HIV产生逆转录酶的唯一途径，逆转录酶是病毒复制的关键酶。因此，通过基础研究阐明TYW 1的作用机制对于理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。 TYW 1被发现具有C-X3-C-X2-C基序，这是自由基SAM超家族的特征。该家族中的酶利用独特的[4Fe-4S]簇来还原性切割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”的未知性质，该52-dA自由基如何催化TYW 1中的N1-甲基鸟嘌呤修饰尚不清楚。 因此，该建议致力于揭示底物“X”，并消除这一重要酶的机理阐明中的最后障碍。化学，生物化学，光谱和酶动力学方法将在我们的实验方法。本研究将利用从不同来源表达的TYW 1酶。此外，tRNAPhe包含通过化学合成制备的m1 G或m1 G类似物也将用于我们的研究。这些实验将使我们能够揭示底物“X”的性质。揭示的“X”结构可以进一步阐明TYW 1的反应机理，这将在本提案以及R21赠款期后的未来研究中进行测试。 公共卫生相关性：TYW 1酶的功能障碍导致苯丙氨酸转移RNA的37位碱基Y（一种鸟嘌呤衍生物）的缺失，这随后使HIV能够产生逆转录酶，这是病毒复制的关键酶。TYW 1的机理研究受到一个未知底物“X”的阻碍，本项目致力于揭示其本质，消除TYW 1催化研究中的主要障碍。更好地了解TYW 1可能最终使其诱导并恢复HIV感染细胞中的Y生物合成，为对抗艾滋病提供新的方法。

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.