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

细菌和人类一样，需要铁才能发挥最佳功能。对于许多细菌来说，铁必须从宿主生物体中窃取，通常是通过使用被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被称为NIS合成酶的未被研究的蛋白质家族。该项目旨在更好地了解这一蛋白质家族，因为它们具有显着的独特化学行为，具有在同一目标上多次进行化学催化的不同寻常的能力(迭代作用)，以及一种前所未有的结构性质。该项目的直接目标是建立实验方法来量化和描述靶标的结合、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被研究的领域，而且有助于更广泛地了解迭代蛋白。这个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。原核生物利用称为铁载体的小分子螯合剂清除铁，这种小分子螯合剂是通过一种新的肽键形成化学方法制成的。一种与细菌毒力日益相关的铁载体合成机制，使用非核糖体多肽铁载体非依赖性合成酶(NIS合成酶)。在这个家族的一个子类别中，存在另外一个显著的动力学行为，由此可以在同一底物上催化多个键的形成(迭代)。迭代活性与广泛的底物特异性相关，但这一点从未被描述或量化。本项目将描述这一新型NIS合成酶家族中重复蛋白的结构、功能和弹性。功能上重要的残基将通过定点突变和使用合成的底物类似物来鉴定。将确定配体和辅因子结合的热力学以及重复底物结合时结构的变化。将进行动力学分析，以量化和比较迭代和单键形成。该项目由生物科学局分子和细胞生物科学司分子生物物理组提供支持。

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.