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 在细菌和蛋白质上安装翻译后修饰（PTM）的酶是不可或缺的 细胞功能，例如生物活性肽天然产物的形成和酶的激活 对细菌适应氧气限制环境的重要性很重要。了解PTM的过程 形成可以告知新型肽疗法的工程和细菌的方法 感染中宿主环境的定殖。 S-腺苷-L-甲硫代（ADOMET）自由基酶的产生 改变目标居住的功能的许多PTM。 Adomet自由基酶执行 对大分子底物上的氧敏感，位点选择性的自由基化学，但一种结构理解 他们如何完成这种令人印象深刻的化学反应已落后于Admet自由基的分析 酶超家族，没有完全可视化的完整的自由基酶 - 蛋白质复合物。目的 该建议包括两种修改特异性Cα的Adomet自由基酶的结构表征 其大底物中的氨基酸：1）具有肽底物的Admet自由基分配酶和2） 丙酮酸与其伴侣PFL的复合物形成裂解酶活性酶（PFL-AE）。分配酶不可逆地转换 L-氨基酸到核糖体肽内的D-氨基酸，从而改变了最终构象和影响 它的生物活性。确定一个分配酶如何将底物定位在指定的多个失误 残留物将需要结构洞察力。 X射线晶体学将用于检查Adomet的相互作用 具有肽底物的自由基二聚糖。 PFL-AE在PFL上形成了催化性必不可少的糖基自由基 从丙酮酸和COA形成形式和乙酰辅酶A。 PFL-AE如何接触PFL和糖基自由基 从PFL-AE活动部位到埋入的PFL活性位点的过渡仍有待阐明。 X射线 晶体学和电子显微镜将用于确定与PFL复合物中PFL-AE的结构。 两种系统的结构分析将为建筑所需的相互作用提供急需的见解 一种蛋白质复合物，该蛋白质复合物进行位点选择性氧敏感的自由基产生化学。