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霉素E492m的生物合成和成熟。微霉素是细菌产生并释放的抗生素肽，用于抑制或杀死邻近不同种类的细菌。MccE492m在翻译后被铁螯合铁载体/盐螯合素修饰。这种修饰允许微霉素与附近细菌细胞上的儿茶酚酸铁载体受体蛋白结合，从而提供更有效的摄取。此外，产铁载体的细菌是毒性最强的细菌之一，因此，MccE492m的生产为某些细菌菌株提供了一种策略，以击败它们最具竞争力的邻居。mce492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，我们将克隆、过表达MccE492基因簇表达的蛋白，并在体外鉴定其活性和底物特异性。特别的重点将放在理解MceC（一种推定的c -糖基转移酶）和MceD（一种推定的酯酶）的反应性上，这两种酶显示出氨基酸同源性，它们与负责产烟碱的iroA基因簇的蛋白质具有同源性。MccE492m翻译后修饰的详细机制研究也将进行。这种修饰是值得注意的，因为它涉及在糖基化的铁载体和MccE492肽的c端丝氨酸残基之间形成酯连接。这些研究将考虑MccE492基因簇的蛋白质，包括Mcel，一种假定的酰基转移酶，以及其他一些功能未知的蛋白质，并确定它们的作用。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讨。特别感兴趣的是确定识别是否需要整个微霉素肽或仅c端或丝氨酸残基。如果催化体系能够在糖基化的铁载体与全长mce492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途径，可能成为有用的抗生素。最后，结合荧光标记和延时共聚焦显微镜，观察mce492m被摄取到原核和真核细胞中，并确定是否进入细胞质。这些生化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mce492m生物合成背后的基本生物化学原理，将为开发针对有毒铁载体产生和革兰氏阴性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.