Structural Characterization of AdoMet Radical Enzyme-Catalyzed Posttranslational Modifications in Bacterial Anaerobic Metabolism

细菌厌氧代谢中 AdoMet 自由基酶催化的翻译后修饰的结构表征

基本信息

批准号：
10057221
负责人：
Emily Ulrich
金额：
$ 6.53万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10057221
关键词：
Acetyl Coenzyme A Acids Activase Active Sites Address Adopted Amino Acids Anabolism Anaerobic Bacteria Bacteria Binding Cell physiology Chemistry Coenzyme A Complex Cryoelectron Microscopy Crystallization DNA biosynthesis Drug Targeting Electron Microscopy Engineering Environment Enzyme Activation Enzymes Fermentation Formulation Future Goals Growth Homologous Gene Hydrocarbons Hydrogen Individual Infection Investigation Length Lyase Metabolism Methionine Methods Modification Molecular Conformation Molecular Sieve Chromatography Molecular Weight Multiple Bacterial Drug Resistance Natural Products Negative Staining Outcome Outcome Study Oxygen Pathogenicity Pathway interactions Peptides Positioning Attribute Post-Translational Protein Processing Process Production Proteins Pyruvate Reaction Ribosomes Roentgen Rays S-Adenosylmethionine Sampling Site Specific qualifier value Specificity Structure Substrate Specificity System X-Ray Crystallography bacterial metabolism bioactive natural products cytotoxic dimer enzyme mechanism enzyme substrate complex epimerase epimerization experimental study formate acetyltransferase activating enzyme host colonization insight member monomer novel novel therapeutics peptide drug prospective protein complex screening

项目摘要

Project Summary Enzymes that install posttranslational modifications (PTMs) on bacterial peptides and proteins are integral in cellular functions such as the formation of bioactive peptide natural products and the activation of enzymes important for bacterial adaptation to oxygen-limited environments. Understanding the process of PTM formation can inform on the engineering of novel peptide therapeutics and on the methods of bacterial colonization of host environments in infection. S-adenosyl-L-methionine (AdoMet) radical enzymes produce numerous PTMs that change the functionality of the targeted residue(s). AdoMet radical enzymes perform oxygen-sensitive, site-selective radical chemistry on macromolecular substrates, yet a structural understanding of how they accomplish this impressive chemistry has lagged behind in the analysis of the AdoMet radical enzyme superfamily, with no complete AdoMet radical enzyme-protein complex fully visualized. The aims of this proposal include structural characterization of two AdoMet radical enzymes that modify the Cα of specific amino acids within their large substrates: 1) an AdoMet radical epimerase with a peptide substrate and 2) pyruvate formate lyase activase (PFL-AE) in complex with its partner PFL. The epimerase irreversibly converts L-amino acids to D-amino acids within a ribosomal peptide, thus altering the final conformation and influencing its bioactivity. Determining how one epimerase positions substrate to perform multiple turnovers at specified residues will require structural insight. X-ray crystallography will be used to examine interactions of AdoMet radical epimerases with peptide substrates. PFL-AE forms the catalytically essential glycyl radical on PFL to make formate and acetyl-CoA from pyruvate and CoA. How PFL-AE contacts PFL and how the glycyl radical transitions from the PFL-AE active site to the buried PFL active site remain to be elucidated. X-ray crystallography and electron microscopy will be used to determine structures of PFL-AE in complex with PFL. Structural analysis of both systems will provide much needed insight into interactions required for construction of a protein complex that performs site-selective oxygen-sensitive radical-generating chemistry.

项目概要在细菌肽和蛋白质上安装翻译后修饰 (PTM) 的酶是细菌中不可或缺的一部分。细胞功能，例如生物活性肽天然产物的形成和酶的激活了解 PTM 过程对于细菌适应缺氧环境非常重要。形成可以为新型肽治疗剂的工程设计和细菌的方法提供信息 S-腺苷-L-甲硫氨酸 (AdoMet) 自由基酶在感染中定植于宿主环境中。许多 PTM 可以改变目标残基的功能。大分子底物上的氧敏感、位点选择性自由基化学，但结构理解他们如何完成这种令人印象深刻的化学反应的研究落后于 AdoMet 自由基的分析酶超家族，没有完整的酶 AdoMet 自由基-蛋白质复合物的目的完全可视化。该提案包括两种 AdoMet 自由基酶的结构表征，这些酶可以修饰特定的 Cα 其大底物内的氨基酸：1) 具有肽底物的 AdoMet 自由基差向异构酶和 2) 丙酮酸甲酸裂解酶激活酶 (PFL-AE) 与其伴侣 PFL 复合，差向异构酶不可逆地转化。核糖体肽内的 L-氨基酸转变为 D-氨基酸，从而改变最终构象并影响确定一种差向异构酶如何定位底物以在指定的情况下进行多次转换。残基需要结构洞察力，将用于检查 AdoMet 的相互作用。自由基差向异构酶与肽底物 PFL-AE 在 PFL 上形成催化必需的甘氨酰自由基。从丙酮酸和 CoA 生成甲酸和乙酰辅酶 A PFL-AE 如何接触 PFL 以及甘氨酰自由基如何。从 PFL-AE 活性位点到埋藏的 PFL 活性位点的转变仍有待阐明。晶体学和电子显微镜将用于确定 PFL-AE 与 PFL 复合物的结构。对两个系统的结构分析将为构建所需的交互提供急需的洞察执行位点选择性氧敏感自由基生成化学的蛋白质复合物。