Single Molecule Studies of Class II Lantibiotic Synthetases

描述（由申请人提供）：抗生素是核糖体合成的，翻译后修饰的肽天然产物，通常具有抗菌活性。抗生素通常通过抑制细菌肽聚糖生物合成中几个高度保守的步骤和/或通过破坏细胞壁和质膜的完整性来起作用。由于l抗生素靶向细菌细胞活力的重要组成部分，它们避免了常见的细菌耐药模式，并已被证明是对抗几种危险的革兰氏阳性人类病原体（如耐甲氧西林金黄色葡萄球菌（MRSA）和耐万古霉素肠球菌）的有效抗生素。认识到l抗生素作为耐药细菌病原体替代疗法的潜力，在过去十年中，对这些肽天然产物的发现和生物合成重新产生了兴趣。这项工作导致了许多抗生素生物合成基因簇的发现和几种生物合成酶的生化表征。所有lantipeptide共同的翻译后修饰（PTMs）包括肽底物（LanA）中丝氨酸和苏氨酸残基的atp依赖脱水，分别形成脱氢丙氨酸（Dha）和脱氢丁氨酸（Dhb）残基，然后半胱氨酸残基在分子内攻击Dha/Dhb残基，形成硫代氨酸（Lan）和甲基硫代氨酸（MeLan）硫醚环。通常，最终产物中存在多个Lan/MeLan环，值得注意的是，对于一些l抗生素，单个生物合成酶（LanM）安装所有这些ptm。由于这些系统中固有的动力学复杂性，各种ptm的时间以及LanM选择特定脱水和环化模式的分子机制在很大程度上是未知的。这一信息对于成功操纵这些酶作为催化剂来设计药物开发中的新型肽结构至关重要。本提案的长期目标是阐明LanM催化的动力学基础，并确定控制底物特异性的系统的动力学和结构特征。为了实现这一目标，将开发几种创新的单分子荧光测定方法，以前所未有的细节对LanM/LanA系统进行动力学表征。这些实验将为多步骤LanM催化反应的各个子步骤提供动力学信息，揭示翻译后修饰事件模式中存在（或不存在）异质性，并定义LanM和LanA之间动态蛋白-蛋白相互作用在确定首选PTM反应途径中的作用。本文开发的分析系统和方法可以应用于许多LanM/LanA系统，以揭示每个系统的一般和独特的催化特征，以及其他肽修饰天然产物生物合成系统的动力学研究。

DESCRIPTION (provided by applicant): Lantibiotics are ribosomally-synthesized, post-translationally modified peptide natural products that often exhibit antimicrobial activity. Lantibiotics typically function by inhibiting several highly conserved steps in bacterial peptidoglycan biosynthesis and/or by disrupting cell wall and plasma membrane integrity. Due to the fact that they target such essential components of bacterial cell viability, lantibiotics hae avoided common modes of bacterial resistance and have proven to be useful antibiotics against several dangerous Gram- positive human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci. Recognizing the potential of lantibiotics as alternative treatments for drug resistant bacterial pathogens, a renewed interest in the discovery and biosynthesis of these peptide natural products has blossomed over the past decade. This work has led to the discovery of many lantibiotic biosynthetic gene clusters and to the biochemical characterization of several of the biosynthetic enzymes. The post-translational modifications (PTMs) that are common to all lantipeptides include the ATP-dependent dehydration of serine and threonine residues in the peptide substrate (LanA) to form dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, respectively, followed by intramolecular attack of cysteine residues onto the Dha/Dhb residues to form lanthionine (Lan) and methyl lanthionine (MeLan) thioether rings. Usually, multiple Lan/MeLan rings are present in the final product and remarkably, for some lantibiotics, a single biosynthetic enzyme (LanM) installs all of these PTMs. Due to the inherent kinetic complexity in these systems, the molecular mechanisms underlying the timing of the various PTMs and the selection of specific dehydration and cyclization patterns by LanM are largely unknown. This information is expected to be critical for successful manipulation of these enzymes as catalysts to engineer novel lantipeptide structures in drug development efforts. The long term goal of this proposal is to elucidate the kinetic basis for LanM catalysis and to identify kinetic and structura features of the system that govern substrate specificity. To achieve this goal, several innovative single molecule fluorescence assays will be developed to kinetically characterize LanM/LanA systems in unprecedented detail. These assays will provide kinetic information for individual sub-steps in the multi-step LanM-catalyzed reaction, reveal the presence (or absence) of heterogeneity in the pattern of post-translational modification events, and define the role of dynamic protein-protein interactions between LanM and LanA in determining the preferred PTM reaction pathway. The assay systems and methods of analysis to be developed herein can be applied to many LanM/LanA systems to uncover the general and unique catalytic features of each system, as well as towards kinetic studies of other peptide-modifying natural product biosynthetic systems.