Examination of the Molecular Properties Underlying the Mechanism, Structure, and Specificity of LanB Enzymes Involved in Lanthipeptide Biosynthesis

 描述（由申请人提供）：生物医学研究界面临的最紧迫挑战之一在于开发新型抗菌剂，作为对抗耐药性病原体进化的手段。因此，货车der Donk的研究集中在羊毛硫肽的研究上，羊毛硫肽是一类核糖体合成和后修饰的肽（RiPP）天然产物，其已被证明具有作为抗生素的显著潜力。事实上，抗生素羊毛硫肽，乳链菌肽，已被用于 美国食品工业近半个世纪以来，很少有抗生素耐药性的报告。这些天然产物的名称来源于其结构内存在的羊毛硫氨酸环，其通过半胱氨酸硫醇与成熟羊毛硫肽内的脱氢丙氨酸和脱氢丁酸残基缀合而形成。最近已经发现，LanBs（在一些羊毛硫肽中负责产生脱氢丙氨酸和脱氢丁酸残基的酶）依赖于谷氨酰-tRNAGlu以谷氨酰化并随后从丝氨酸和苏氨酸消除羟基。不幸的是，很少有人知道的酶的机制，这发生或如何调节。因此，通过使用同位素标记的辅因子，非反应性底物模拟物，诱变和酶动力学分析，本文提出的研究旨在研究LanB活性的化学和结构特征。要实现的具体目标包括确定NisB的谷氨酸转移的机制和化学选择性，谷氨酸化和消除活性位点的结构阐明，以及确定tRNA辅因子识别的分子机制。通过更好地了解这些特性，我们的总体目标是更好地了解羊毛硫肽的生物合成，以便为新型抗生素和治疗化合物的开发提供信息。

 DESCRIPTION (provided by applicant): One of the most pressing challenges to the biomedical research community lies in the development of novel antimicrobials as a means of combating the evolution of antibiotic-resistant pathogens. As such, research in the van der Donk is focused on the study of lanthipeptides, a class of ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products, which have demonstrated significant potential as antibiotics. Indeed, the antibiotic lanthipeptide, nisin, has been used in the U.S. food industry for almost half a century with very few reports of antibiotic resistance. These natural products derive their name from the presence of lanthionine rings within their structure, which are formed through the conjugation of cysteine thiols to dehydroalanine and dehydrobutyrine residues within the maturing lanthipeptide. It has recently been found that LanBs, the enzymes responsible for the generation of dehydroalanine and dehydrobutyrine residues in some lanthipeptides, rely upon glutamyl-tRNAGlu in order to glutamylate and subsequently eliminate the hydroxyl-group from serine and threonine. Unfortunately, little is known about the enzymatic mechanisms by which this occurs or how it is regulated. Therefore, through the use of isotopically labeled cofactors, non-reactive substrate mimics, mutagenesis, and enzyme kinetics analysis, the research proposed herein is designed to examine the chemical and structural features that underlie LanB activity. The specific goals to be achieved include the determination of the mechanism and chemoselectivity of glutamate transfer by NisB, the structural elucidation of the glutamylation and elimination active sites, and the molecular mechanisms that determine tRNA cofactor recognition. Through a better understanding of these properties, our overall objective is to better understand lanthipeptide biosynthesis in order to inform the development of novel antibiotic and therapeutic compounds.