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

项目总结/摘要（Mark内斯比特） 自由基S-腺苷甲硫氨酸（SAM）酶（RSE）超家族负责催化 各种各样的不寻常的和困难的化学反应，这些反应对生物体的生存至关重要 从细菌到人类。RSE通过氧化还原活性[4Fe-4S]簇通过三个 半胱氨酸残基（通常来自CX 3CX 2C序列）。RSE催化反应的初始步骤， SAM与[4Fe-4S]簇结合并被还原生成Ado·（5 '-脱氧腺苷自由基）， 这是所有RSE的共同点。然而，尽管有许多结构上的相似之处， 步骤RSE能够催化多种化学转化， 修饰、金属蛋白簇组装和辅因子生物合成。两个RSE参与肽 修饰是PqqE和Mftc。PqqE催化氧化还原酶的生物合成中的C-C键形成步骤。 辅因子PQQ。MftC催化MftA中C-末端酪氨酸残基的氧化脱羧 肽作为建议的氧化还原辅因子mycofactocin的生物合成的一部分。研究机制 这些基因相关的酶能够催化截然不同的化学反应， 对RSE在自然界中的各种功能有价值的见解。 计划的研究将询问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 酪氨酸残基由MfTC催化。连续波EPR和脉冲方法，如ENDOR，将能够 深入探索潜在的自由基有机和有机金属中间体的性质。使用非自然 设计用于稳定潜在自由基中间体并掺入肽类似物中的氨基酸 衬底将有助于这些实验。其他光谱方法，如快速冷冻淬火57 Fe 穆斯堡尔光谱学将提供补充EPR研究的数据，允许观察所有 翻转条件下样品中的铁核。这些实验产生的数据将有助于进一步研究 对自由基SAM酶催化的各种生物反应的机理理解， 提供了在催化下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，并将提供强有力的 光谱基础知识的机制，通过这种不同的家庭酶 完成了它们所适应的独特生物学角色。

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.