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

• 批准号: 10246524
• 负责人: Emily Ulrich
• 金额: $ 5.67万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246524
• 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

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.

项目摘要 在细菌肽和蛋白质上安装翻译后修饰（PTM）的酶是细菌蛋白质中不可或缺的。 细胞功能，如生物活性肽天然产物的形成和酶的活化 对细菌适应氧气有限的环境很重要。了解PTM流程 形成可以告知新的肽治疗剂的工程和细菌的方法， 在感染中宿主环境的定殖。S-腺苷-L-甲硫氨酸（NAMet）自由基酶产生 许多改变目标残基功能的PTM。蛋氨酸自由基酶执行 对大分子底物的氧敏感性、位点选择性自由基化学， 他们是如何完成这种令人印象深刻的化学反应的， 酶超家族，没有完全可视化的完整的Met自由基酶-蛋白质复合物。的目的 这一建议包括两种修饰特异性的Cα的α-Met自由基酶的结构表征， 在其大底物内的氨基酸：1）具有肽底物的α-Met自由基差向异构酶和2） 丙酮酸甲酸裂解酶活化酶（PFL-AE）与其配偶体PFL复合。差向异构酶不可逆地将 核糖体肽内的L-氨基酸转化为D-氨基酸，从而改变最终构象并影响 其生物活性。确定一种差向异构酶如何定位底物以在指定的温度下进行多次翻转 残留物需要结构洞察。X射线晶体学将被用来检查的相互作用， 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基自由基， 从丙酮酸和CoA中制备甲酸和乙酰CoA。PFL-AE如何接触PFL以及甘氨酰自由基如何 从PFL-AE活性位点到掩埋的PFL活性位点的转变仍有待阐明。x射线 晶体学和电子显微镜将用于确定PFL-AE与PFL复合物的结构。 对这两个系统的结构分析将为施工所需的相互作用提供急需的见解 一种蛋白质复合物，它能进行位点选择性的氧敏感性自由基生成化学反应。