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: 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
• 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

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 生物活性多肽天然产物的形成和酶的激活等细胞功能 对细菌适应氧气有限的环境很重要。理解PTM的过程 形成可以为新的肽疗法的工程和细菌的方法提供信息 在感染中寄主环境的定植。S-腺苷-L-蛋氨酸(ADOMet)自由基酶产生 改变目标残基官能度的大量PTM(S)。ADOMet自由基酶表现 大分子底物上氧敏感的、位置选择性的自由基化学，但对结构的理解 他们是如何完成这一令人印象深刻的化学的，在对ADOMet激进分子的分析中落后了 酶超家族，没有完整的Adobe Met自由基酶-蛋白复合体完全可见。的目标是 该建议包括两种ADOMet自由基酶的结构特征，这两种酶可以改变特定的Cα 其大底物中的氨基酸：1)带有多肽底物的Adobe Met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形成复合体。差向异构酶不可逆地转化为 L-氨基酸转变为核糖体内的D-氨基酸，从而改变了最终的构象并影响 它的生物活性。确定一个差向异构酶如何定位底物以在指定的时间执行多次翻转 残留物将需要对结构的洞察。X射线结晶学将被用来研究ADOMet的相互作用 带有多肽底物的自由基异构酶。PFL-AE在PFL上形成催化必需的甘氨酸根 丙酮酸和辅酶A合成甲酸盐和乙酰辅酶A。PFL-AE如何与PFL接触以及甘氨酸根是如何 从PFL-AE活性中心到埋藏的PFL活性中心的转变还有待阐明。X射线 结晶学和电子显微镜将被用来确定PFL-AE与PFL络合物的结构。 对这两个系统的结构分析将为构建所需的相互作用提供亟需的洞察 指一种蛋白质复合体，它执行位置选择性的氧敏感自由基生成化学。