Characterization of YcaO-Dependent Natural Product Biosynthetic Pathways

YcaO 依赖性天然产物生物合成途径的表征

• 批准号: 10389609
• 负责人: Douglas Alan Mitchell
• 金额: $ 7.09万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389609
• 关键词: Address; Anabolism; Attention; Bioinformatics; Biology; C-terminal; Chemicals; Data; Doctor of Philosophy; Environment; Enzymatic Biochemistry; Enzymes; Family; Family member; Funding; Generations; Genomics; Knowledge; Logic; Medicine; Modification; N-terminal; Natural Products; Pathway interactions; Peptides; Positioning Attribute; Proteins; Reaction; Reporting; Ribosomes; Speed; Structure; Structure-Activity Relationship; Sulfur; Vertebral column; base; cycloaddition; interest; member; microbial; novel; pyridine

Our group is broadly interested in the chemical biology of natural products with a strong focus on genomics- based discovery, biosynthetic mechanistic enzymology, and determination of structure-activity relationships and mode of action. Beyond their historical impact on medicine, natural products have inspired generations of syn- thetic chemists and provided the necessary chemical probes to illuminate fundamental aspects of biology. One natural product family that has received increased attention over the past several years are the ribosomally synthesized and post-translationally modified peptides (RiPPs). While there are over 30 distinct structural clas- ses of RiPP natural products reported, they are united by a common biosynthetic logic: a precursor peptide, typically composed of an N-terminal leader and a C-terminal core, is ribosomally produced. The leader region contains motifs that are recognized by the modification enzymes and the core region is where the modifications take place. Upon maturation, the leader region is often removed prior to cellular export. The current project focuses on natural product biosynthetic pathways that encode a member of the YcaO superfamily. During the original funding period, we showed that YcaO enzymes were responsible for the ATP- dependent activation of the peptide backbone to yield azoline heterocycles from Cys, Ser, and Thr residues of the core peptide. During the current funding period, we discovered that two additional reaction types are cata- lyzed by YcaO enzymes: thioamidation and macrolactamidation of the peptide backbone. No fewer than five classes of RiPPs are now known to utilize a member of the YcaO superfamily, namely the linear azol(in)e- containing peptides, thiopeptides, cyanobactins, bottromycins, and thioviridamides. Despite a wealth of knowledge, we can only predict the modification type of approximately one-third of the YcaO superfamily. Our bioinformatics analysis suggests that several new reaction types remain to be discovered. For this renewal project, we tackle several outstanding questions with respect to YcaO-dependent natural product biosynthesis. Aim I focuses on the structurally and enzymatically intriguing thiopeptide RiPP class. Aim IA will overcome biosynthetic bottlenecks with respect to substrate tolerance in order to establish the elusive structure-activity relationships and generate advanced biosynthetic intermediates that will enable the study of late-stage transformations found within the class. Aim IB will determine the enzymatic mechanism and substrate scope of the class-defining [4+2]-cycloaddition and establish why some are pyridine-forming while others are dehydropiperidine-forming. Aim II focuses on peptide backbone thioamidation, in particular, deciphering the func- tion of the TfuA partner protein and a novel desulfurase/lysase involved in mobilizing sulfur from Cys. Lastly, Aim III characterizes divergent YcaO family members that appear in unique genomic contexts to discover new reac- tions catalyzed by the superfamily. Our preliminary data, rich environment, and strong investigative team place us in an ideal position to address these aims.

我们的团队对天然产品的化学生物学广泛感兴趣，并强烈关注基因组学- 基于发现、生物合成机械酶学和结构-活性关系的确定以及 行动模式。除了对医学的历史影响外，天然产品还激励了一代又一代人-- 并提供了必要的化学探针，以阐明生物学的基本方面。一 在过去几年中受到越来越多关注的天然产品家族是核糖体 人工合成和翻译后修饰的多肽(RIPP)。虽然有30多个不同的结构类别- 报道的RIPP天然产物的SE，它们由一个共同的生物合成逻辑联系在一起：前体多肽， 通常由N-末端前导和C-末端核心组成，以核糖体形式产生。领头羊地区 包含由修饰酶识别的基序，核心区是修饰的位置 去做吧。成熟后，前导区通常在细胞输出之前被移除。 目前的项目重点是编码YcaO成员的天然产物生物合成途径 超级大家庭。在最初的资助期，我们发现YcaO酶负责ATP- 依赖激活多肽骨架以从半胱氨酸、丝氨酸和苏氨酸残基生成唑啉杂环 核心肽。在目前的资助期内，我们发现另外两种反应类型是Cata- YCaO酶裂解：肽骨架的硫代酰胺化和大内酰胺化。不少于五个 现在已知的RIPP类利用YcaO超家族的一种成员，即线性天青(In)e- 含有多肽、硫代多肽、氰基巴拉菌素、肉毒菌素和硫代维达胺。尽管有大量的 据我们所知，我们只能预测大约三分之一的YcaO超家族的修饰类型。我们的 生物信息学分析表明，仍有几种新的反应类型有待发现。 对于这个更新项目，我们解决了与依赖YcaO的Natural有关的几个悬而未决的问题 产品生物合成。目的研究硫肽Ripp类化合物的结构和酶学特性。目标 IA将克服有关底物耐受性的生物合成瓶颈，以建立难以捉摸的 结构-活性关系，并产生先进的生物合成中间体，使研究 在类中发现的后期转换。AIM IB将确定酶的作用机制和底物 定义类的[4+2]-环加成反应的范围，并确定为什么有些是吡啶形成的，而另一些是 脱氢哌啶的形成。AIM II侧重于肽骨架的硫代酰胺化，特别是对其功能的破译。 TfuA配对蛋白和一种新的参与从半胱氨酸中动员硫的脱硫酶/裂解酶的表达。最后，目标 III描述了出现在独特基因组环境中以发现新反应的不同的YcaO家族成员。 在超级家族的催化下。我们的初步数据、丰富的环境和强大的调查团队所在 美国处于实现这些目标的理想位置。