RUI: Structural and Functional Substrate Binding in Iterative Non-ribosomal peptide synthesis Independent Synthesis (NIS) Enzyme DesD

RUI：迭代非核糖体肽合成中的结构和功能底物结合独立合成 (NIS) 酶 DesD

• 批准号: 1716986
• 负责人: Katherine Hoffmann
• 金额: $ 19.51万
• 依托单位: California Lutheran University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1716986&HistoricalAwards=false
• 关键词: RUI; Structural; Functional; Substrate; Binding

Bacteria, like humans, require iron to function optimally. For many bacteria that iron must be stolen from the host organism, often through the use of small chemicals called siderophores that bind iron. The most virulent bacteria create siderophores through a series of steps that involve one or more of an understudied family of proteins called NIS Synthetases. This project is designed to better understand this family of proteins due to their remarkable unique chemical behavior and the unusual ability to enact chemical catalysis multiple times on the same target (iterative action) as well as a never before seen structural property. The immediate goal for this project is to establish experimental methods to quantify and describe binding of targets, the structure and iterative behavior of the protein. The long-term goal of the research is to describe the mechanism of NIS synthetases in detail, to contribute understanding not only to this understudied field, but also of iterative proteins more broadly. This project will provide training and education primarily to members of underrepresented groups and first generation college students.Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis mechanism, increasingly associated with bacterial virulence, uses Non-ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases). Within a sub-category in this family, an additional remarkable kinetic behavior exists whereby the formation of multiple bonds may be catalyzed on the same substrate (iterative.) The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. This project will characterize the structure, function, and elasticity of the iterative proteins in this novel family of NIS sythetases. Functionally important residues will be identified by site-directed mutagenesis and the use of synthetically prepared substrate analogs. Thermodynamics of ligand and cofactor binding and changes in structure with iterative substrate binding will be determined. Kinetic assays will be performed to quantify and compare iterative vs. single bond formation. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.

细菌和人类一样，需要铁来发挥最佳功能。对于许多细菌来说，铁必须从宿主生物体中窃取，通常是通过使用称为铁载体的小化学物质来结合铁。毒性最强的细菌通过一系列步骤产生铁载体，这些步骤涉及一种或多种未充分研究的蛋白质家族，称为NIS合成酶。该项目旨在更好地了解这个蛋白质家族，因为它们具有显着的独特化学行为和在同一目标上多次实施化学催化的不寻常能力（迭代作用）以及前所未有的结构特性。该项目的近期目标是建立实验方法来量化和描述靶点的结合，蛋白质的结构和迭代行为。这项研究的长期目标是详细描述NIS合成酶的机制，不仅有助于理解这一未充分研究的领域，而且有助于更广泛地理解迭代蛋白质。 该项目将主要为代表性不足的群体成员和第一代大学生提供培训和教育。原核生物使用称为铁载体的小分子螯合剂来螯合铁，通过一种新的化学肽键形成。一种类型的铁载体合成机制，越来越多地与细菌毒力相关，使用非核糖体肽铁载体独立合成酶（NIS合成酶）。在这个家族的一个子类中，存在一个额外的显着的动力学行为，由此可以在同一底物上催化形成多个键（迭代）。迭代活性与广泛的底物特异性相关，但这从未被描述或量化。这个项目将描述这个新的NIS合成酶家族中迭代蛋白的结构、功能和弹性。功能重要的残基将通过定点诱变和使用合成制备的底物类似物来鉴定。热力学的配体和辅因子结合和结构的变化与迭代底物结合将被确定。将进行动力学测定，以定量和比较迭代与单键形成。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理学小组的支持。

项目成果

Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family

• DOI: 10.1021/acs.biochem.9b00899
• 发表时间: 2020-09-22
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Hoffmann, Katherine M.;Goncuian, Eliana S.;Orion, Iris W.
• 通讯作者: Orion, Iris W.