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

基本信息

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
项目类别：
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

项目摘要

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，重点关注基因组学- 基于发现、生物合成机制酶学以及结构-活性关系的确定和行动方式。除了对医学的历史影响之外，天然产品还启发了一代又一代的科学家主题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。一在过去几年中受到越来越多关注的天然产物家族是核糖体合成的和翻译后修饰的肽（RiPP）。虽然有超过 30 种不同的结构类别据报道，RiPP 天然产物的系列，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 端前导序列和 C 端核心组成，由核糖体产生。领导者地区包含被修饰酶识别的基序，核心区域是修饰的地方发生。成熟后，前导区域通常在细胞输出之前被去除。当前的项目重点关注编码 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，我们证明 YcaO 酶负责 ATP- 肽主链的依赖性激活，从Cys、Ser和Thr残基产生唑啉杂环核心肽。在当前的资助期间，我们发现另外两种反应类型是催化的通过 YcaO 酶裂解：肽主链的硫代酰胺化和大环内酰胺化。不少于五个现在已知 RiPP 类别利用 YcaO 超家族的成员，即线性 azol(in)e- 含有肽、硫肽、蓝菌素、bottromycins 和 thioviridamides。尽管拥有丰富的据了解，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，几种新的反应类型仍有待发现。对于这个更新项目，我们解决了与 YcaO 依赖的天然物质有关的几个悬而未决的问题。产物生物合成。目标 I 重点关注结构和酶学上令人感兴趣的硫肽 RiPP 类。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，这将使研究班级内发现的后期转变。目标 IB 将确定酶促机制和底物定义类的 [4+2]-环加成的范围，并确定为什么有些是吡啶形成的，而另一些是形成脱氢哌啶。目标 II 重点关注肽骨架硫代酰胺化，特别是破译其功能 TfuA 伴侣蛋白和一种新型脱硫酶/裂解酶参与从 Cys 中调动硫。最后，瞄准 III 描述了不同的 YcaO 家族成员，它们出现在独特的基因组环境中，以发现新的反应由超家族催化的。我们的初步数据、丰富的环境、强大的调查团队我们处于实现这些目标的理想位置。