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

项目摘要 普遍存在的多重耐药细菌和新抗菌剂发现缓慢的组合 治疗学威胁着破坏过去一个世纪的许多全球公共卫生进步。的主要关注点 范德唐克实验室正在了解天然产物的生物合成和作用机理 抗菌活性。特别是，该实验室对一类被称为羊硫肽的分子感兴趣 具有强大的抗菌活性，可能是对抗抗生素耐药性的有用模式。几个 羊毛肽正在进行临床试验，用于治疗耐药细菌感染，而抗生素羊毛肽 几十年来，Nisin一直被用于食品工业，以对抗食源性病原体。第I类羊毛肽 脱水酶，通称为LANB，在羊毛肽和其他生物合成中起关键作用。 通过将丝氨酸/苏氨酸残基转化为脱氢丙氨酸/脱氢丁碱，将天然产物分类。范德那辆 DONK实验室最近证明了LANB利用带电的tRNA来谷氨酰化丝氨酸/苏氨酸侧链醇， 导致一种酯被消除以形成脱水氨基酸。蓝宝石的晶体结构 揭示了一种模块化的结构，其中谷氨酰化和消除活性被定位为分离 域名。有趣的是，在基因组数据库中发现的许多酶完全没有消除结构域。 因此，这些较短的LanB(SLanB)酶可能具有不同的生物合成逻辑，并可能先前产生 未知的多肽天然产物。为了研究这些基因簇，一种旨在 建议使用最新的分析工具来确定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，高分辨质量 光谱学、稳定同位素标记、异源表达和生物信息学。在这些方面进行培训 方法将极大地扩展申请者的技能集。具体目标包括确定最佳文化 在本土生产商中分离sLanB衍生天然产物的条件，调查氨基的去向 来自带电tRNA的酸性加合物，以及在不同生物合成环境中是否存在sLanB酶 共享一个通用的机制。这些目标包括鉴定两个不同sLanB基因的最终产物。 并对其功能进行评估。鉴于类LanB酶参与了一系列有效和 在临床和商业上的成功治疗，sLABS的产品可能具有宝贵的生物学价值 活动。此外，了解(S)LABS催化的反应和初步的机理表征 可能会扩大生物信息学工具箱，用于对新的天然产物进行基因组挖掘。

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.