Biosynthesis of hypermodified gaunosines.

项目摘要。转移RNA(TRNA)的转录后处理涉及许多 功能上不同的事件对tRNA的成熟至关重要。核苷修饰的现象是 也许是这些事件中最引人注目的，并导致了大量的结构变化 核苷。在tRNA中发现的两种最引人注目的修饰核苷是7-去氮鸟苷 在翻译和RNA稳定中具有假定作用的衍生物Queuosine和Archosine， 分别进行了分析。虽然这些核苷在进化上是相关的，但它们分离在不同的结构域中； 队列蛋白在细菌和真核生物中普遍存在，而考古蛋白只存在于古细菌中。这个 7-去氮嘌呤结构广泛存在于生物学中，存在于多种天然产物中。 例如来自链霉菌的抗肿瘤抗生素托伊卡霉素、圣维菌素和杀瘤素。这个 对这些去氮嘌呤的生物合成途径知之甚少，这一事实阻碍了功能的发挥 学习。数百个测序基因组的可获得性现在允许识别基因和 使用比较基因组学方法的途径。这种方法被用来发现五种新的酶 在Queuosine和Arcosine的从头生物合成中，以及潜在的其他7-去氮嘌呤的生物合成中 代谢物。值得注意的是，这一途径仅限于原核生物，其中一些新发现的酶 似乎催化了生物中前所未有的化学作用。这项计划的长远目标是， 阐明7-地氮嘌呤在原核生物中的生物合成和代谢。这样做的具体目的是 建议1)阐明这些新的酶在队列蛋白和 始祖生物合成途径导致共同前体7-氰基-7-氰基-7- 去氮鸟嘌呤，2)启动对这些修饰核苷更广泛代谢的研究，以及3) 研究其中一种酶的机制和结构，一种新型的丁腈氧化还原酶。这 提案带来了一系列生物信息学、遗传学、生化和化学方法来解决这个问题。 阐明了7-去氮鸟嘌呤修饰核苷的生物合成。这一新途径的研究和应用 这些组成酶将为阐明7-去氮嘌呤的生物学提供前所未有的途径。 新陈代谢及其控制。 关联性。7-去氮尿嘧啶的途径是微生物所特有的，许多构成 酶是潜在的新的抗菌目标。此外，其中一种酶是一种新型的丁腈 氧化还原酶在工业生物催化中的应用。

Project Summary. The post-transcriptional processing of transfer RNA (tRNA) involves a number of functionally distinct events essential for tRNA maturation. The phenomenon of nucleoside modification is perhaps the most remarkable of these events, and results in a wealth of structural changes to the canonical nucleosides. Two of the most remarkable modified nucleosides found in tRNA are the 7-deazaguanosine derivatives queuosine and archaeosine, which have putative roles in translation and RNA stabilization, respectively. While evolutionarily related, these nucleosides are segregated within separate Domains; queuosine is ubiquitous among Bacteria and Eukarya, while archaeosine is only present in the Archaea. The 7-deazapurine structure in general is widespread in biology, where it is found in a variety of natural products such as the antitumor antibiotics toyocamycin,sangivamycin, and tubercidin from Streptomyces. The biosynthetic pathways to these deazapurines are poorly understood, a fact that has stymied functional studies. The availability of hundreds of sequenced genomes now allows the identification of genes and pathways using a comparative genomics approach. This approach was used to discover five new enzymes in the de novo biosynthesis of queuosine and archaeosine, and potentially of other 7-deazapurine metabolites. Notably, this pathway is limited to prokaryotes, and some of these newly discovered enzymes appear to catalyze chemistry unprecedented in biology. The long-term objectives of this project are to , elucidate the biosynthesis and metabolism of 7-deazapurines in prokaryotes. The specific aims of this proposal are 1) to elucidate the role of these new enzymes in the early steps in the queuosine and archaeosine biosynthetic pathways leading to the formation of the common precursor 7-cyano-7- deazaguanine, 2) to initiate studies into the broader metabolism of these modified nucleosides, and 3) to investigate the mechanism and structure of one of these enzymes, a novel nitrile oxidoreductase. This proposal brings an ensemble of bioinformatic, genetic, biochemical, and chemical approaches to the problem of elucidating the biosynthesis of 7-deazaguanine modified nucleosides. The study of this new pathway and the constituent enzymes will provide unprecedented access to elucidating the biology of 7-deazapurine metabolism and its manipulation. Relevance. The pathway to 7-deazapurines is unique to microorganisms, and many of the constitute enzymes are potentially new antibacterial targets. Furthermore, one of the enzymes is a novel nitrile oxidoreductase that may have applications in industrial biocatalysis.