Characterization of novel cobalamin-dependent radical SAM methylase via substrate identification in C. difficile

通过艰难梭菌中的底物鉴定表征新型钴胺素依赖性自由基 SAM 甲基化酶

• 批准号: 10315912
• 负责人: Amy Elizabeth Solinski
• 金额: $ 6.6万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10315912
• 关键词: Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Proteins; Binding; Biochemical; Biochemistry; Biogenesis; Bioinformatics; Biological; Biological Assay; Biology; C-terminal; Carbon; Characteristics; Chemistry; Clostridium difficile; Cobalamin; Communicable Diseases; Complex; Crystallization; Data; Development; Disease; Enzymes; Escherichia coli; Family; Future; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Human; Investigation; Iron; Knowledge; Life; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Methylation; Methyltransferase; Microbiology; Pathway interactions; Physiological; Play; Protein Binding Domain; Protein Biosynthesis; Proteins; Proteomics; Radiolabeled; Reaction; Research; Research Personnel; Resolution; Ribosomes; Roentgen Rays; Role; S-Adenosylhomocysteine; Solubility; Structure; Subgroup; Sulfur; System; Techniques; Testing; Therapeutic; Time; Transcobalamins; Work; base; design; disorder prevention; enzyme structure; enzyme substrate; experimental study; global health; human disease; human pathogen; insight; methyl group; novel; protein expression; protein purification; therapeutically effective; uptake

Project Summary Biological methylation plays an integral role in human disease by regulating pathways important for homeostasis, disease prevention and in some scenarios, can even promote disease. For example, in bacterial ribosome systems, seemingly simple methylations can result in the development of antibiotic resistance to current antibiotics. For a long time, methylations were known to be completed by an SN2 transfer of the methyl group from S-Adenosyl methionine (SAM) to a nucleophilic heteroatom. Then, about two decades ago, it was discovered that iron-sulfur [4Fe-4S] clusters had the ability to promote radical cleavage of SAM and form 5’-deoxyadenosyl 5’-radical (5’dA•), which can promote radical methylation of previously unactivated carbon centers! Sequencing capabilities have highlighted the vast presence of these radical SAM methylases (RSMs) in biology, but it is still unknown what unique chemistries these enzymes are capable of, and more importantly, how RSMs contribute to human health. The Booker lab at Penn State has been working on characterizing RSMs that are dependent on cobalamin (Cbl) as an intermediate methyl carrier during the methylation reaction. Recently, we have discovered a novel subgroup of Cbl-dependent RSMs that contain an atypical Cbl-binding protein domain. Previously unannotated, and referred to as a domain of unknown function (DUF512), this domain differs from the canonical Cbl-binding domains, as it is C-terminal to the RS motif. Using bioinformatics, we have identified ~4000 proteins that contain DUF512, including one from the important human pathogen Clostridioides difficile. We expect the C. difficile DUF512 (cdDUF512) enzyme to perform a radical based methylation on an unactivated carbon center of a protein substrate. Preliminary work, has shown that cdDUF512 is connected to ribosome maturation, and we hypothesize that cdDUF512 methylates EngA, an essential GTPase that stabilizes the 50S subunit of the ribosome. Ribosomal protein synthesis is a significant antibiotic target, thus highlighting the importance of the proposed work as we uncover cdDU512’s mechanism in C. difficile. We propose the investigation of cdDUF512, to decipher the structural importance of the novel DUF512 domain, identify the biological substrate of these enzymes, and understand the biological importance in C. difficile, while creating a roadmap for future RSM annotation. First, the x-ray crystal structure for cdDUF512 will be solved to decipher its important binding characteristics. Second, we will examine cdDUF512’s connection to ribosome maturation with a combined biochemical and proteomic approach. Lastly, we will use a radiolabeling technique, which was developed in the Booker lab, to track the methylation in cellular lysate to identify the biological substrate. In all, we hope to progress the field of radical SAM chemistry forward, while deciphering mechanisms that will contribute to future antibiotic development.

项目概要 生物甲基化通过调节重要途径在人类疾病中发挥着不可或缺的作用 用于体内平衡、疾病预防，在某些情况下甚至可以促进疾病。例如，在 细菌核糖体系统，看似简单的甲基化可以导致抗生素的开发 对当前抗生素的耐药性。长期以来，甲基化被认为是通过 SN2 转移完成的 将甲基从 S-腺苷甲硫氨酸 (SAM) 转变为亲核杂原子。然后，大约两 几十年前，人们发现铁硫[4Fe-4S]簇具有促进自由基裂解的能力 SAM 并形成 5'-脱氧腺苷 5'-自由基 (5'dA•)，可促进 SAM 的自由基甲基化 以前未活化的碳中心！测序能力凸显了这些的广泛存在 生物学中的自由基 SAM 甲基化酶 (RSM)，但仍不清楚这些酶的独特化学性质 更重要的是，RSM 能够为人类健康做出贡献。 宾夕法尼亚州立大学的布克实验室一直致力于表征依赖的 RSM 钴胺素（Cbl）作为甲基化反应过程中的中间甲基载体。最近，我们有 发现了 Cbl 依赖性 RSM 的新亚组，其中含有非典型 Cbl 结合蛋白 领域。该结构域以前未注释，被称为未知功能结构域 (DUF512) 与典型的 Cbl 结合域不同，因为它位于 RS 基序的 C 端。利用生物信息学，我们 已鉴定出约 4000 种含有 DUF512 的蛋白质，其中一种来自重要的人类病原体 艰难梭菌。我们期望艰难梭菌 DUF512 (cdDUF512) 酶能够执行基于自由基的操作。 蛋白质底物未活性碳中心的甲基化。初步工作表明 cdDUF512 与核糖体成熟有关，我们假设 cdDUF512 甲基化 EngA， 稳定核糖体 50S 亚基的必需 GTP 酶。核糖体蛋白质的合成是 重要的抗生素靶标，因此在我们发现 cdDU512 时强调了拟议工作的重要性 艰难梭菌中的机制。 我们建议对 cdDUF512 进行研究，以破译新型 DUF512 的结构重要性 域，识别这些酶的生物底物，并了解其生物学重要性 C. difficile，同时为未来的 RSM 注释创建路线图。一、cdDUF512的x射线晶体结构 将被解决以破译其重要的结合特征。其次，我们将检查 cdDUF512 结合生化和蛋白质组学方法与核糖体成熟的联系。最后，我们将使用一个 布克实验室开发的放射性标记技术，用于追踪细胞裂解物中的甲基化 识别生物底物。总之，我们希望推动自由基 SAM 化学领域的发展， 同时破译有助于未来抗生素开发的机制。