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: 10474316
• 负责人: Amy Elizabeth Solinski
• 金额: $ 6.76万
• 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/10474316
• 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 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 对于体内稳态，预防疾病和某些情况，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发展 对当前抗生素的抗性。长期以来，已知甲基被SN2转移完成 从甲基甲二氨氨酸（SAM）到核嗜素杂原子的甲基的甲基。然后，大约两个 几十年前，发现铁硫[4FE-4S]簇具有促进自由基裂解的能力 SAM和形式的5'-脱氧丁糖基5'-RADICAL（5'DA•），可以促进自由基甲基化 以前未活化的碳中心！测序功能强调了这些功能的大量存在 生物学中的自由基SAM甲基酶（RSM），但这些酶是什么独特化学 RSM有能力，更重要的是，RSM如何为人类健康做出贡献。 宾夕法尼亚州的布克实验室一直在努力表征依赖的RSM 在甲基化反应过程中，在钴胺素（CBL）上是中间甲基载体。最近，我们有 发现了一个新型CBL依赖性RSM的亚组，其中包含非典型CBL结合蛋白 领域。以前未经通知，被称为未知函数的域（DUF512），该域 与RS基序的C末端，与规范CBL结合域不同。使用生物信息学，我们 已经确定了约4000种含有DUF512的蛋白质，其中一种来自重要人类病原体 梭状芽胞杆菌艰难梭菌。我们期望艰难梭菌DUF512（CDDUF512）酶执行基于自由基的酶 在蛋白质底物的未活化碳中心上的甲基化。初步工作表明 CDDUF512连接到核糖体成熟，我们假设CDDUF512甲基化ENGA，A 稳定核糖体50S亚基的必需GTPase。核糖体蛋白合成是一种 当我们发现CDDU512时，重大抗生素靶标，因此强调了拟议工作的重要性 艰难梭菌的机制。 我们建议CDDUF512的投资，以破译新颖的DUF512的结构重要性 域，确定这些酶的生物学底物，并了解生物学重要性 艰难梭菌，同时为未来的RSM注释创建路线图。首先，CDDUF512的X射线晶体结构 将解决以破译其重要的结合特征。第二，我们将检查CDDUF512的 与核糖体成熟的连接，结合生化和蛋白质组学方法。最后，我们将使用 在布克实验室开发的辐射标记技术，以跟踪细胞裂解物中的甲基化 确定生物基底物。总的来说，我们希望进步激进的Sam化学领域， 而解密的机制将有助于未来的抗生素发展。