Mechanistic Explorations of Microcin E492m Biosynthesis and Maturation

描述(申请人提供)：本研究方案的长期目标是了解微霉素E492m的生物合成和成熟。微球蛋白是细菌产生和释放的抗菌肽，用于抑制或杀死不同物种的邻近细菌。MccE492m是用铁螯合铁质载体/沙莫胆碱部分翻译后修饰的。这种修饰允许微球素与附近细菌细胞上的儿茶素铁载体受体蛋白结合，从而提供更有效的摄取。此外，产生铁载体的细菌是毒力最强的细菌之一，因此MccE492m的生产为某些细菌菌株提供了一种击败最具竞争力的邻居的策略。MccE492m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，MccE492基因簇表达的蛋白质将被克隆、过度表达，并在体外鉴定其活性和底物特异性。将特别强调了解MCEC和MCed的反应性，MCEC是一种假定的C-糖基转移酶，MCed是一种假定的酯酶，它们显示出与负责Salmochelin产生的iroA基因簇的蛋白质的氨基酸同源性。还将对MccE492M的平移后改装进行详细的机械调查。这种修饰是值得注意的，因为它涉及糖基化的铁载体和MccE492肽的C-末端丝氨酸残基之间形成的酯键。这些研究将考虑MccE492基因簇的蛋白质，包括推测的酰基转移酶McEL和其他几种功能未知的蛋白质，并确定它们的作用(S)。一旦这种翻译后修饰的机制细节被阐明，底物识别和特异性将被探索。特别感兴趣的是确定识别是否需要整个微蛋白多肽或只需要C末端或丝氨酸残基。如果催化系统能够在糖基化的铁载体和全长MccE492肽以外的底物之间形成酯键，该装置将为制备铁载体-毒素结合物提供一种新的方法，这可能是有用的抗生素。最后，结合荧光标记和延时共聚焦显微镜将被用来显示MccE492m摄取到原核和真核细胞，并确定是否发生细胞质进入。这些生化研究与公共卫生特别相关，因为抗生素耐药性是一个严重的世界性问题。揭示MccE492m生物合成背后的基础生物化学将为开发针对产生铁载体和革兰氏阴性杆菌的毒力强的铁载体和革兰氏阴性细菌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.