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.

项目总结