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: 10220046
• 负责人: Douglas Alan Mitchell
• 金额: $ 34.15万
• 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/10220046
• 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; 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.

我们的团队对天然产物的化学生物学广泛感兴趣，重点关注基因组学- 基于发现，生物合成机制酶学，并确定结构-活性关系， 行动方式。除了它们对医学的历史影响外，天然产品还激发了几代人的合成， 他们是化学家，并提供了必要的化学探针来阐明生物学的基本方面。一 在过去的几年里，受到越来越多关注的天然产物家族是核糖体 合成的和后修饰的肽（RIPPs）。虽然有30多个不同的结构类- 据报道，RiPP天然产物的种类，它们通过共同的生物合成逻辑结合在一起：前体肽， 通常由N-末端前导序列和C-末端核心组成，是核糖体产生的。领导者区域 含有被修饰酶识别的基序，并且核心区域是修饰酶 发生。成熟后，前导区通常在细胞输出之前被去除。 目前的项目重点是编码YcaO成员的天然产物生物合成途径 超家族在最初的资助期间，我们发现YcaO酶负责ATP- 依赖性活化肽骨架以从Cys、Ser和Thr残基产生唑啉杂环， 核心肽在目前的资助期间，我们发现另外两种反应类型是催化剂， 通过YcaO酶裂解：肽骨架的硫代酰胺化和大环内酰胺化。不少于五 现在已知一类RIPP利用YcaO超家族的成员，即线性azol（in）e-， 含有肽、硫肽、蓝藻菌素、bottromycins和硫代病毒酰胺。尽管有丰富的 根据我们的知识，我们只能预测大约三分之一的YcaO超家族的修饰类型。我们 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，我们解决了几个悬而未决的问题，关于YcaO依赖的自然 产物生物合成。目的研究具有结构和酶学性质的硫肽类RiPP。目的 IA将克服底物耐受性方面的生物合成瓶颈，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，这将使研究 在班级中发现的后期转换。目的研究酶的作用机理和底物 定义类别的[4+2]-环加成的范围，并确定为什么有些是吡啶形成的，而另一些是 脱氢哌啶形成。目的II侧重于肽骨架硫代酰胺化，特别是，破译功能， TfuA伴侣蛋白和一种新的脱硫酶/裂解酶参与动员硫从半胱氨酸。最后，Aim III表征了出现在独特基因组背景中的不同YcaO家族成员，以发现新的基因表达。 由超家族催化的反应我们的初步数据、丰富的环境和强大的调查团队 我们处于实现这些目标的理想位置。