Mechanistic Explorations of Microcin E492m Biosynthesis and Maturation

描述（由申请人提供）：本研究计划的长期目标是了解微菌素E492 m的生物合成和成熟。微菌素是细菌产生和释放的抗生素肽，以抑制或杀死不同物种的邻近细菌。MccE 492 m是用铁螯合铁载体/salmochelin部分进行后修饰的。这种修饰允许微菌素与附近细菌细胞上的儿茶酚铁载体受体蛋白结合，这提供了更有效的摄取。此外，产生铁载体的细菌是毒性最强的细菌之一，因此MccE 492 m的产生为某些细菌菌株提供了击败其最具竞争力的邻居的策略. MccE 492 m组装的逻辑目前尚不清楚，揭示这一过程的细节将有助于为开发新的抗生素药物提供策略。为了实现这一目标，MccE 492基因簇表达的蛋白质将被克隆，过表达，并在体外表征其活性和底物特异性。特别强调将被放置在理解的反应性MceC，一个假定的C-糖基转移酶，和MceD，一个假定的酯酶，显示氨基酸同源性与负责salmochelin生产的iroA基因簇的蛋白质。还将对MccE 492 m翻译后修饰进行详细的机制研究。这种修饰是显著的，因为它涉及在糖基化的铁载体和MccE 492肽的C-末端丝氨酸残基之间形成酯键。MccE 492基因簇的蛋白质，包括Mcel，一种假定的酰基转移酶，和其他几种功能未知的蛋白质将在这些研究中被考虑，并确定其作用。一旦阐明了这种翻译后修饰的机制细节，将探索底物识别和特异性。特别感兴趣的是确定是否需要整个小菌素肽或仅C-末端或丝氨酸残基进行识别。如果该催化体系能在糖基化铁载体和全长MccE 492肽以外的底物之间形成酯键，则该机制将为制备铁载体-毒素缀合物提供新的方法，这可能是有用的抗生素。最后，将采用荧光标记和延时共聚焦显微镜的组合来可视化MccE 492 m摄取到原核和真核细胞中，并确定是否发生细胞质进入。这些生物化学研究与公共卫生特别相关，因为抗生素耐药性是世界范围内的一个严重问题。揭示MccE 492 m生物合成背后的基本生物化学将为开发用于有毒铁载体生产和革兰氏阴性细菌菌株的新型抗生素提供新的见解。

DESCRIPTION (provided by applicant): The long-term objective of this research proposal is to understand the biosynthesis and maturation of microcin E492m. Microcins are antibiotic peptides that bacteria produce and release to inhibit or kill neighboring bacteria of different species. MccE492m is post-translationally modified with an iron-chelating siderophore/salmochelin moiety. This modification allows the microcin to bind to catecholate siderophore receptor proteins on nearby bacterial cells, which affords more efficient uptake. Additionally, siderophore- producing bacteria are among the most virulent, and MccE492m production therefore provides a strategy for certain bacterial strains to defeat their most competitive neighbors. The logic of MccE492m assembly is currently unknown and unveiling the details of this process will help provide strategies for the development of new antibiotic drugs. Toward this goal, proteins expressed by the MccE492 gene cluster will be cloned, over-expressed and their activities and substrate specificities characterized in vitro. Particular emphasis will be placed on understanding the reactivity of MceC, a putative C-glycosyltransferase, and MceD, a putative esterase, that show amino acid homology with proteins of the iroA gene cluster responsible for salmochelin production. A detailed mechanistic investigation of the MccE492m post-translational modification will also be undertaken. This modification is remarkable since it involves formation of an ester linkage between a glycosylated siderophore and a C-terminal serine residue of the MccE492 peptide. Proteins of the MccE492 gene cluster, including Mcel, a putative acyltransferase, and several other proteins of unknown function will be considered in these studies and their role(s) established. Once the mechanistic details of this post- translational modification are elucidated, substrate recognition and specificity will be probed. Of particular interest is to determine if the entire microcin peptide or only the C-terminus or serine residue is required for recognition. If the catalytic system can form ester linkages between the glycosylated siderophore and substrates other than the full-length MccE492 peptide, the machinery will offer a new approach for preparing siderophore-toxin conjugates, which may be useful antibiotics. Lastly, a combination of fluorescence labeling and time-lapse confocal microscopy will be employed to visualize MccE492m uptake into prokaryotic and eukaryotic cells and to ascertain if cytoplasmic entry occurs. These biochemical studies are of particular relevance to public health because antibiotic resistance is a serious problem worldwide. Unveiling the fundamental biochemistry behind MccE492m biosynthesis will provide new insights for the development of novel antibiotics for virulent siderophore-producing and gram-negative bacterial strains.