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

检查参与羊毛硫肽生物合成的 LanB 酶的机制、结构和特异性的分子特性

• 批准号: 9525480
• 负责人: Ian Roger Bothwell
• 金额: $ 0.07万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-16 至 2019-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9525480
• 关键词: Active Sites; Address; Amides; Amino Acids; Amino Acyl Transfer RNA; Anabolism; Antibiotic Resistance; Antibiotics; Biochemical; Biomedical Engineering; Biomedical Research; Catalysis; Catalytic Domain; Chemicals; Communities; Complex; Cyclization; Cysteine; Data; Dehydration; Development; Diagnostic radiologic examination; Drug resistance; Enzyme Kinetics; Enzymes; Evolution; Food Industry; Foundations; Future; Generations; Glutamates; Glutamic Acid; Glutamic Acid-Specific tRNA; Goals; Health; Human; Hydro-Lyases; Isotope Labeling; Light; Mediating; Methods; Molecular; Mutagenesis; Names; Natural Products; Nature; Nisin; Peptides; Pharmacologic Substance; Process; Production; Property; Reaction; Reporting; Research; Resistance development; Ribosomes; Role; Serine; Side; Site; Specific qualifier value; Specificity; Stable Isotope Labeling; Structure; Sulfhydryl Compounds; Techniques; Therapeutic; Threonine; Transfer RNA; Work; analog; antimicrobial; antimicrobial drug; carboxylate; cofactor; combat; dehydroalanine; dehydrobutyrine; design; experimental study; hydroxyl group; insight; lanthionine; member; mutant; novel; pathogen; peptide structure; public health relevance

 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.

 描述（由申请人提供）：生物医学研究界最紧迫的挑战之一在于开发新型抗菌药物作为对抗抗生素抗性病原体进化的手段，因此，范德唐克的研究重点是。对 lanthipeptides 的研究，一类核糖体合成和翻译后修饰肽 (RiPP) 天然产物，已显示出作为抗生素的巨大潜力。乳链菌肽，已被用于 近半个世纪以来，这些天然产品在美国食品工业中几乎没有出现抗生素耐药性的报道，其名称来源于其结构中存在的羊毛硫氨酸环，这些环是通过半胱氨酸硫醇与内部的脱氢丙氨酸和脱氢丁氨酸残基结合而形成的。最近发现 LanBs 是一些酶，负责在某些物质中产生脱氢丙氨酸和脱氢丁氨酸残基。 lanthipeptides 依赖于谷氨酰-tRNAGlu 来谷氨酰化并随后消除丝氨酸和苏氨酸中的羟基，不幸的是，人们对这种情况发生的酶促机制或如何通过使用同位素标记进行调节知之甚少。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检查 LanB 活性背后的化学和结构特征。要实现的具体目标包括确定 NisB 谷氨酸转移的机制和化学选择性、谷氨酰化和消除活性位点的结构阐明以及决定 tRNA 辅因子识别的分子机制。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。