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

 描述(申请人提供)：生物医学研究界面临的最紧迫的挑战之一是开发新型抗菌剂，作为对抗耐药病原体进化的一种手段。因此，van der Donk的研究集中在羊毛肽的研究上，这是一类核糖体合成和翻译后修饰多肽(RIPP)的天然产物，已显示出巨大的抗生素潜力。事实上，抗生素羊毛肽Nisin已经被用于 近半个世纪以来，美国食品业几乎没有抗生素耐药性的报告。这些天然产物的名字来源于它们结构中存在的兰硫环，这些环是通过半胱氨酸硫醇与成熟的兰硫肽中的脱氢丙氨酸和脱氢丁碱残基结合而形成的。最近发现，在一些羊毛肽中负责产生脱氢丙氨酸和脱氢丁碱残基的酶LABS依赖于谷氨酰-tRNAGlu来谷氨化，随后从丝氨酸和苏氨酸中消除羟基。不幸的是，人们对这种情况发生的酶机制或它是如何调节的知之甚少。因此，通过使用同位素标记的辅因子、非反应底物模拟物、诱变和酶动力学分析，本文提出的研究旨在检查支撑LAB活性的化学和结构特征。要实现的具体目标包括确定NisB转移谷氨酸的机制和化学选择性，阐明谷氨酸化和消除活性部位的结构，以及决定tRNA辅因子识别的分子机制。通过更好地了解这些性质，我们的总体目标是更好地了解羊硫肽的生物合成，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.