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.

项目摘要 生物甲基化通过调节重要的途径在人类疾病中起着不可或缺的作用 对于体内平衡，疾病预防，在某些情况下，甚至可以促进疾病。例如在 在细菌核糖体系统中，看似简单的甲基化可以导致抗生素的发展 对现有抗生素的耐药性。很长一段时间以来，已知甲基化是通过SN 2转移完成的 从S-腺苷甲硫氨酸（SAM）的甲基基团到亲核杂原子。然后，大约两个 几十年前，人们发现铁-硫[4Fe-4S]团簇具有促进自由基裂解的能力 5 ′-脱氧腺苷5 ′-自由基（5 'dA·）可促进SAM的自由基甲基化， 以前没有活性炭的中心！测序能力突出了这些基因的广泛存在， 自由基SAM甲基化酶（RSM）在生物学中，但它仍然是未知的，这些酶是什么独特的化学 更重要的是，RSMs如何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描述依赖于 钴胺素（Cbl）作为甲基化反应期间的中间甲基载体。最近我们 发现了一个新的Cbl依赖性RSM亚群，其中含有非典型Cbl结合蛋白 域以前未注释，并被称为未知功能域（DUF 512），该域 与典型的Cbl结合结构域不同，因为它是RS基序的C末端。利用生物信息学，我们 已经鉴定了约4000种含有DUF 512的蛋白质，其中包括一种来自重要的人类病原体的蛋白质 艰难梭菌我们期待C。艰难梭菌DUF 512（cdDUF 512）酶进行基于自由基的 在一些实施方案中，所述方法包括在蛋白质底物的未活性碳中心上的甲基化。初步工作表明， cdDUF 512与核糖体成熟有关，我们假设cdDUF 512甲基化EngA， 稳定核糖体50 S亚基的必需GT3。核糖体蛋白质合成是一种 重要的抗生素靶点，从而突出了我们发现cdDU 512的拟议工作的重要性 机制在C.很难 我们建议对cdDUF 512进行研究，以破译新型DUF 512的结构重要性 结构域，确定这些酶的生物底物，并了解生物学的重要性， C.困难，同时为未来的RSM注释创建路线图。首先，cdDUF 512的X射线晶体结构 将被解决，以破译其重要的绑定特性。第二，我们将检查cdDUF 512的 用生物化学和蛋白质组学的方法研究核糖体成熟的联系。最后，我们将使用 放射性标记技术，这是在布克实验室开发的，用于跟踪细胞裂解物中的甲基化 来识别生物基质。总之，我们希望推进自由基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。