Single Molecule Studies of Class II Lantibiotic Synthetases

描述(申请人提供)：抗生素是核糖体合成的、翻译后修饰的多肽天然产品，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的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发挥作用。由于它们针对细菌细胞生存的这些基本成分，抗生素避免了细菌耐药的常见模式，并已被证明是对几种危险的革兰氏阳性人类病原体的有效抗生素，如甲氧西林耐药金黄色葡萄球菌(MRSA)和万古霉素耐药肠球菌。认识到抗生素作为抗药性细菌病原体替代疗法的潜力，在过去十年中，人们对这些多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，并对几种生物合成酶进行了生化表征。翻译后修饰(PTM)是所有多肽共有的，包括依赖于ATP的多肽底物(LANA)上的丝氨酸和苏氨酸残基的脱水，分别生成脱氢丙氨酸(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，形成羊硫氨酸(LAN)和甲基羊硫氨酸(Melan)硫代醚环。通常，最终产品中存在多个Lan/Melan环，并且值得注意的是，对于某些抗生素，单个生物合成酶(LAMM)安装所有这些PTM。由于这些体系固有的动力学复杂性，各种PTM的时间选择以及LAMM选择特定的脱水和环化模式的分子机制在很大程度上是未知的。这一信息有望对成功地将这些酶作为催化剂在药物开发工作中设计新的反肽结构至关重要。这项建议的长期目标是阐明LAMM催化的动力学基础，并确定控制底物专一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分析方法，以前所未有的详细程度对LAMM/LANA体系进行动力学表征。这些分析将提供多步骤LAMM催化反应中各个子步骤的动力学信息，揭示翻译后修饰事件模式中异质性的存在(或不存在)，并确定LAMM和LANA之间动态蛋白质-蛋白质相互作用在确定首选PTM反应途径中的作用。这里将要开发的分析系统和方法可以应用于许多LAMM/LANA系统，以揭示每个系统的一般和独特的催化特性，以及用于其他多肽修饰的天然产物生物合成系统的动力学研究。 公共卫生相关性：需要新的抗生素化合物来减轻耐药细菌病原体对人类健康造成的令人望而生畏的治疗。抗生素是一类很有前途的多肽天然产物，可以避免常见的细菌耐药机制。这一建议旨在获得对构成抗生素的酶的详细机理的了解，希望更好地操纵它们的活性，以用于工程设计或设计合成具有新的或改进的生物活性的新化合物。

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. PUBLIC HEALTH RELEVANCE: New antibiotic compounds are needed to mitigate the daunting treat to human health posed by drug resistant bacterial pathogens. Lantibiotics are a promising class of peptide natural products that avoid common bacterial resistance mechanisms. This proposal seeks to gain a detailed mechanistic understanding of the enzymes that construct lantibiotics, in hopes of better manipulating their activity for the engineering or designed synthesis of novel compounds with new or improved biological activity.