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: 10457879
• 负责人: Douglas Alan Mitchell
• 金额: $ 32.62万
• 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/10457879
• 关键词: Actinobacteria class; Address; Alkenes; Amidines; Anabolism; Antibiotics; Archaea; Attention; Biochemical; Bioinformatics; Biological Assay; Biology; C-terminal; Carbon; Chemicals; Chemistry; Clinical; Data; Deltaproteobacteria; Development; Doctor of Philosophy; Environment; Enzymatic Biochemistry; Enzymes; Evaluation; Family; Family member; Funding; Gene Cluster; Generations; Genes; Genetic; Genomics; Gram-Positive Bacteria; Growth; Human; Hydro-Lyases; In Vitro; Isotopes; Kinetics; Knowledge; Ligation; Logic; Longevity; Mass Spectrum Analysis; Mechanics; Medicine; Microbe; Modeling; Modification; Molecular; Mutation Analysis; N-terminal; Natural Products; Nature; Nucleotides; Pathway interactions; Peptides; Positioning Attribute; Post-Translational Protein Processing; Property; Proteins; Reaction; Reporting; Research; Resolution; Ribosomes; Role; Source; Speed; Structure-Activity Relationship; Sulfides; Sulfur; Thiamine; Thiazoles; Thioamides; Variant; Vertebral column; adenylate; analog; base; cofactor; cycloaddition; enolase; interest; member; microbial; novel; pyridine; quantum; recruit; success; thioether

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.

我们的小组对天然产物的化学生物学广泛感兴趣，重点是基因组学 - 基于发现的发现，生物合成机械酶学以及确定结构活性关系和 行动方式。除了对医学的历史影响外，天然产品还激发了几代人的合成 Thetic化学家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，人们受到越来越受到关注的天然产品家族是核糖体 合成和翻译后修饰的肽（RIPP）。虽然有30多个不同的结构clas- 报道的RIPP天然产品的SES是由常见的生物合成逻辑结合在一起的：一种前体肽， 通常由N末端引导者和C末端核心组成，是核糖体产生的。领导区 包含通过修饰酶识别的基序，而核心区域是修饰的地方 发生。成熟后，通常在细胞出口之前去除领导区域。 当前的项目着重于编码YCAO成员的天然产品生物合成途径 超家族。在原始资金期间，我们表明YCAO酶是ATP-的原因 肽主链的依赖激活以产生来自Cys，Ser和THR残基的二唑杂环 核心肽。在当前的资金期间，我们发现另外两种反应类型是cata- 由YCAO酶溶解：肽主链的硫代和大乳酸化。不少于五 现在已知RIPP类别使用YCAO超家族的成员，即线性偶氮（IN）E- 含有肽，硫代肽，蓝霉素，肉豆蔻霉素和硫代依他胺。尽管有很多 知识，我们只能预测YCAO超家族的大约三分之一的修改类型。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。 对于这个续签项目，我们解决了有关依赖YCAO的自然的几个杰出问题 产品生物合成。目的我重点关注结构和酶上吸引人的硫代肽RIPP类。目的 IA将克服相对于底物公差的生物合成瓶颈，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，以实现研究 班级中发现的后期转变。 AIM IB将确定酶促机制和底物 定义类别的范围[4+2] -Cycloadition，并确定为什么有些是吡啶形成的，而另一些则是在 脱氢哌啶形成。 AIM II侧重于肽骨架硫代塑性，特别是破译了功能 TFUA伴侣蛋白的TION和一种新型的脱硫酶/裂解酶，参与了Cys的硫。最后，目标 III的特征是在独特的基因组环境中出现的不同YCAO家庭成员，以发现新的recc- 由超家族催化的。我们的初步数据，丰富的环境和强大的调查团队地点 我们处于解决这些目标的理想位置。