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

项目摘要 生物甲基化通过调节重要的途径在人类疾病中发挥着不可或缺的作用 对于动态平衡、疾病预防和在某些情况下，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发展 对目前的抗生素产生抗药性。很长一段时间以来，甲基化都是通过SN2转移来完成的 从S-腺苷甲硫氨酸(SAM)到亲核杂原子的甲基。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切割的能力 和形成5‘-脱氧腺苷5’-自由基(5‘da·)，可促进自由基甲基化 以前的非活性碳中心！测序能力突显了这些基因的广泛存在 生物学中的自由基SAM甲基酶(RSM)，但目前还不清楚这些酶是什么独特的化学作用 能够，更重要的是，RSM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表征依赖于 在甲基化反应中，以钴胺(Cbl)作为中间甲基载体。最近，我们有 发现了一个新的依赖Cbl的RSM亚群，它含有一个非典型的Cbl结合蛋白 域。以前未加注释的，称为未知函数的域(DUF512)，此域 不同于典型的Cbl结合域，因为它是RS基序的C末端。利用生物信息学，我们 已经确定了大约4000种含有DUF512的蛋白质，其中包括一种来自人类重要病原体的蛋白质 艰难梭状芽胞杆菌我们预计艰难梭菌DUF512(CdDUF512)酶将执行基于自由基的 蛋白质底物非活性碳中心上的甲基化。初步工作表明， CdDUF512与核糖体成熟有关，我们假设cdDUF512甲基化EngA，And。 稳定核糖体50s亚基的必需GTP酶。核糖体蛋白合成是一种 重要的抗生素靶点，从而突出了拟议工作的重要性，因为我们发现了cdDU512‘S 艰难梭菌的致病机制。 我们建议对cdDUF512进行研究，以破译新的DUF512的结构重要性 结构域，确定这些酶的生物底物，并了解在 艰难梭菌，同时为未来的RSM注释创建了路线图。首先，cdDUF512的x射线晶体结构 将被破解以破译其重要的结合特征。第二，我们将审查cdDUF512的S 生物化学和蛋白质组学相结合的方法与核糖体成熟的联系。最后，我们将使用 由布克实验室开发的用于跟踪细胞裂解物中甲基化的放射性标记技术 以鉴定生物底物。总而言之，我们希望推动激进的SAM化学领域向前发展， 同时破译有助于未来抗生素发展的机制。

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.