Mechanistic studies of peptide modifying radical S-adenosylmethionine enzymes PqqE and MftC

肽修饰自由基S-腺苷甲硫氨酸酶PqqE和MftC的机理研究

• 批准号: 9889972
• 负责人: Mark A Nesbit
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889972
• 关键词: Anabolism; Bacteria; Binding; Biological; Carbon; Cell Nucleus; Chemicals; Collection; Complement; Coupling; Cysteine; Data; Decarboxylation; Electron Nuclear Double Resonance; Enzymes; Family; Foundations; Freezing; Geometry; Glutamates; Human; Individual; Investigation; Iron; Isotope Labeling; Knowledge; Label; Life; Mediating; Metalloproteins; Methods; Modification; Mossbauer Spectroscopy; Nature; Organism; Oxidation-Reduction; PQQ Cofactor; Peptides; Physiologic pulse; Play; Positioning Attribute; Publishing; Reaction; Reporting; Research; Role; S-Adenosylmethionine; Sampling; Spectrum Analysis; Structure; Sulfur; System; Techniques; Testing; Time; Tyrosine; Variant; analog; chemical reaction; cofactor; design; electronic structure; experimental study; formate acetyltransferase activating enzyme; insight; interest; member; oxidation; peptide analog; spectroscopic data; unnatural amino acids

Project Summary/Abstract (Mark Nesbit) The superfamily of radical S-adenosylmethionine (SAM) enzymes (RSEs) are responsible for catalyzing a wide variety of unusual and difficult chemical reactions which are critical for the survival of living organisms from bacteria to humans. RSEs are identified by binding of a redox active [4Fe-4S] cluster through three cysteine residues (typically from a CX3CX2C sequence). The initial step in RSE catalyzed reactions where SAM binds to the [4Fe-4S] cluster and is reduced to generate Ado• (5’-deoxyadenosyl radical) appears to be common to all RSEs. However, despite having many structural similarities and sharing a common initiation step RSEs are able to catalyze a wide variety of chemical transformations and are involved in peptide modification, metalloprotein cluster assembly and cofactor biosynthesis. Two RSEs involved in peptide modification are PqqE and MftC. PqqE catalyzes a C-C bond forming step in the biosynthesis of the redox cofactor PQQ. MftC catalyzes the oxidative decarboxylation of the C-terminus tyrosine residue in the MftA peptide as a part of the biosynthesis of the proposed redox cofactor mycofactocin. Studying the mechanisms by which these genetically related enzymes are able to catalyze vastly different chemical reactions will provide valuable insight into the varied functionality of RSEs in nature. The planned investigation will interrogate the mechanism of the C-C bond forming step in PQQ biosynthesis catalyzed by the radical SAM enzyme PqqE and the oxidative decarboxylation of a C-terminus tyrosine residue catalyzed by MftC. Continuous wave EPR, and pulsed methods such as ENDOR will be able to intimately probe the nature of potential radical organic and organometallic intermediates. The use of non-natural amino acids designed to stabilize potential radical intermediates and incorporated into analogs of the peptide substrates will assist in these experiments. Additional spectroscopic methods such as rapid freeze-quench 57Fe Mössbauer spectroscopy will provide data which complements the EPR studies allowing for observation of all Fe nuclei in a sample under turnover conditions. The data produced by these experiments will help to further the mechanistic understanding of the diverse array of biological reactions catalyzed by radical SAM enzymes and provide additional characterization of physical and electronic structures of the PqqE and MftC under catalytically relevant conditions. Additionally, these methods may be used to study other RSEs and will provide a strong spectroscopic foundation of knowledge about the mechanisms by which this diverse family of enzymes accomplishes the unique biological roles they have adapted to fill.

项目摘要/摘要（Mark Nesbit） 自由基S-腺苷甲硫代（SAM）酶（RSE）的超家族负责催化 各种各样的异常和困难的化学反应对于生物的生存至关重要 从细菌到人类。通过三个氧化还原活性[4FE-4S]群集来识别RSE 半胱氨酸残留物（通常来自CX3CX2C序列）。 RSE催化反应的第一步，其中 SAM与[4FE-4S]簇结合，并减少以生成ADO•（5'-脱氧丁糖基自由基）似乎是 所有RSE共同。但是，尽管有许多结构相似性并共享共同的启动 步骤RSE能够催化多种化学转化，并参与肽 修饰，金属蛋白簇组装和辅因子生物合成。参与肽的两个RSE 修改是PQQE和MFTC。 PQQE在氧化还原的生物合成中催化C-C键形成步骤 cofactor pqq。 MFTC催化MFTA中C末端酪氨酸居住的氧化脱羧 肽作为提出的氧化还原辅因子菌乳素的生物合成的一部分。研究机制 这些一般相关的酶能够催化截然不同的化学反应将提供 对自然界中RSE的各种功能的有价值的见解。 计划的调查将询问PQQ中C-C键形成步骤的机制 由自由基SAM酶PQQE催化的生物合成和C末端的氧化脱羧化 MFTC催化的酪氨酸居住。连续波EPR和脉冲方法（例如Endor）将能够 亲密探测潜在的自由基有机和有机中间体的性质。使用非天然 氨基酸旨在稳定潜在的自由基中间体，并掺入辣椒的类似物中 底物将有助于这些实验。其他光谱法方法，例如快速冻结57FE Mössbauer光谱法将提供完成EPR研究的数据，允许观察所有研究 在周转条件下样品中的Fe核。这些实验产生的数据将有助于进一步 从根治性SAM酶和 提供催化下PQQE和MFTC的物理和电子结构的额外表征 相关条件。此外，这些方法可用于研究其他RSE，并将提供强大的 有关该酶的潜水家族的知识的光谱基础 完成了他们适应填补的独特生物学角色。