Investigation of the role of tRNA-dependent aminoacyl transferases in peptide natural product biosynthesis

项目摘要 普遍存在的多药耐药细菌和新抗菌药物发现的缓慢 治疗学有可能破坏过去世纪的许多全球公共卫生进步。的主要重点 货车德东克实验室正在了解天然产物的生物合成和作用机制， 抗菌活性。特别是，该实验室对一类称为羊毛硫肽的分子感兴趣， 具有有效的抗微生物活性，可能是对抗抗生素耐药性的有用模型。几 lanthipeptide正在临床试验中治疗耐药细菌感染，抗生素lanthipeptide 乳酸链球菌肽在食品工业中用于对抗食源性病原体已有数十年。I类羊毛硫肽 通常称为LanBs的腺苷酸酶在羊毛硫肽以及其他生物活性物质的生物合成中起关键作用。 通过将Ser/Thr残基转化为脱氢丙氨酸/脱氢丁酸来制备天然产物。货车der Donk实验室最近证明LanB利用带电的tRNA使Ser/Thr侧链醇谷氨酰化， 导致酯被消除以形成脱水氨基酸。LanB的晶体结构 揭示了一个模块化的架构，其中谷氨酰化和消除活动是本地化的，以分离 域.有趣的是，在基因组数据库中发现的许多酶完全缺失消除域。 因此，这些短LanB（sLanB）酶可能具有不同的生物合成逻辑，并且可能先前产生 未知肽天然产物。为了研究这些基因簇，一项研究策略旨在 建议使用最新的分析工具确定sLanB酶的产物和生物合成逻辑， 包括细菌培养条件的操作、转录分析、高分辨率质谱分析、 光谱法、稳定同位素标记、异源表达和生物信息学。培训这些 这些方法将大大扩展申请人的技能组合。具体目标包括确定最佳文化 在天然生产者中分离sLanB衍生的天然产物的条件，研究氨基的命运， 酸加合物来源于带电的tRNA，并建立在不同的生物合成环境中的sLanB酶 共享一个通用机制。这些目标包括鉴定两种不同sLanB基因的终产物， 集群，并评估其功能。考虑到LanB样酶参与一系列有效的， 临床上和商业上成功的治疗剂，sLanB的产物可能具有有价值的生物学活性。 活动此外，了解sLanBs催化什么反应和初始机理表征 可以扩展生物信息学工具箱，用于新的天然产物的基因组挖掘。

Project Summary The combination of pervasive multidrug-resistant bacteria and the slowing discovery of new antimicrobial therapeutics threatens to undermine many global public health advances of the past century. A main focus of the van der Donk lab is understanding the biosynthesis and mechanism of action of natural products with antimicrobial activities. In particular, the lab is interested in a class of molecules known as lanthipeptides that have potent antimicrobial activities and may be useful models for combating antibiotic resistance. Several lanthipeptides are in clinical trials to treat drug-resistant bacterial infections, and the antibiotic lanthipeptide nisin has been used for decades in the food industry to combat food-borne pathogens. Class I lanthipeptide dehydratases, generically termed LanBs, play a key role in the biosynthesis of lanthipeptides as well as other classes of natural products by converting Ser/Thr residues into dehydroalanine/dehydrobutyrine. The van der Donk lab recently demonstrated that LanBs utilize charged tRNAs to glutamylate Ser/Thr side chain alcohols, leading to an ester that is eliminated to form the dehydrated amino acid. The crystal structure of a LanB revealed a modular architecture in which the glutamylation and elimination activities are localized to separate domains. Intriguingly, many enzymes found in genome databases are missing the elimination domain entirely. These short LanB (sLanB) enzymes may thus have a different biosynthetic logic and likely produce previously unknown peptide natural products. In order to investigate these gene clusters, a research strategy aimed at determining the products and biosynthetic logic of sLanB enzymes is proposed using the latest analytical tools, including manipulation of bacterial culture conditions, transcriptional analysis, high-resolution mass spectrometry, stable isotope labeling, heterologous expression, and bioinformatics. Training in these approaches will greatly expand the skill set of the applicant. Specific goals include determining optimal culture conditions for isolation of sLanB-derived natural products in a native producer, investigating the fate of amino acid adducts derived from charged tRNAs, and establishing if sLanB enzymes in different biosynthetic contexts share a general mechanism. These aims include identifying the end products of two distinct sLanB gene clusters and evaluating their function. Given the involvement of LanB-like enzymes in a range of potent and clinically and commercially successful therapeutics, the products of sLanBs may have valuable biological activities. Additionally, understanding what reaction(s) sLanBs catalyze and initial mechanistic characterization may expand the bioinformatic toolbox for genome mining for novel natural products.