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Structural determinants of reaction specificity in PLP-dependent arginine oxidases

PLP 依赖性精氨酸氧化酶反应特异性的结构决定因素

基本信息

批准号：
1903899
负责人：
Nicholas Silvaggi
金额：
$ 41.1万
依托单位：
University of Wisconsin-Milwaukee
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1903899&HistoricalAwards=false
关键词：
Structural determinants reaction specificity PLP

项目摘要

A general understanding in biochemistry is that the molecular structure of an enzyme defines its function and, thus, two enzymes having the same or very similar structures (homologs) should catalyze the same or similar chemical reactions. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Nicholas Silvaggi from the University of Wisconsin Milwaukee to determine how homologs of an enzyme from two different organisms produce different products using different chemical steps. The pyridoxal-5'-phosphate-dependent L-arginine deaminase (or MppP) enzyme from the organism Streptomyces wadayamensis (SwMppP) was found by Dr. Silvaggi to react with molecular oxygen to add a hydroxyl group onto the amino acid arginine. The enzyme from Paenibacillus elgii (PeMppP) has a nearly identical structure to SwMppP, but instead creates a new double bond in the middle of the arginine side chain. Both products are ultimately incorporated into secondary metabolites including antibiotics. The lessons learned from these studies help to predict the functions of new proteins uncovered through genomic studies, and to better understand how existing enzyme structures adapt to perform new functions. An outreach program is developed by Dr. Silvaggi to improve the scientific literacy of the public. This program produces short video explanations of scientific questions raised by 3rd and 4th grade students, with the goal of showing how science relates in very intimate ways to their everyday lives.The non-proteinogenic amino acids L-enduracididine (L-End) and 4,5-dehydroarginine (dhArg) are components of bacterially-produced natural products mannopeptimycin and indolmycin, respectively. Both L-End and dhArg are produced by PLP-dependent oxidases. The enzyme from Streptomyces wadayamensis (SwMppP) uses molecular oxygen to install a hydroxyl group at carbon 4 of the L-Arg substrate side chain, while that from Paenibacillus elgii (PeMppP) creates a double bond between carbons 4 and 5 (PeMppP). SwMppP and PeMppP have nearly identical tertiary structures and share identical active site residues. The only structural differences appear to be at the dimer interface, leading to the hypothesis that differences in the quaternary structure impact the shapes of the active sites and thus the binding mode of the L-Arg substrate. A primary question being addressed is how these two structurally similar enzymes synthesize different products through their different chemical mechanisms. Although much is known (or can be inferred) about the mechanisms of SwMppP and PeMppP, neither mechanism has been fully characterized. In this project, the novel alternative substrate 3,4-dehydro-L-arginine is used to bypass the early stages and thus probe the late stages of the catalytic mechanism of SwMppP though stopped-flow and quench-flow kinetics, NMR spectroscopy, and mass spectrometry experiments. A similar strategy is used to study the mechanism of PeMppP. The outcomes of this research will be detailed information on the reaction mechanisms of these two MppP-like proteins and consequently how evolution has modified the Type I aminotransferase fold to perform new catalytic functions. These outcomes expand our knowledge of PLP-dependent enzymes specifically and of enzyme structure-function relationships in general, and improves the accuracy of protein function predictions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

生物化学中的一般理解是，酶的分子结构决定了其功能，因此，具有相同或非常相似结构（同系物）的两种酶应该催化相同或相似的化学反应。有了这个奖项，化学部的生命过程化学计划正在资助来自威斯康星州密尔沃基大学的Nicholas Silvaggi博士，以确定来自两种不同生物的酶的同系物如何使用不同的化学步骤产生不同的产物。Silvaggi博士发现来自生物体Streptomyces wadayamensis（SwMppP）的吡哆醛-5 '-磷酸依赖性L-精氨酸脱氨酶（或MppP）与分子氧反应，在氨基酸精氨酸上添加羟基。来自类芽孢杆菌的酶（PeMppP）与SwMppP具有几乎相同的结构，但在精氨酸侧链的中间产生了一个新的双键。这两种产物最终都被纳入次级代谢产物，包括抗生素。从这些研究中吸取的经验教训有助于预测通过基因组研究发现的新蛋白质的功能，并更好地了解现有酶结构如何适应新功能。Silvaggi博士制定了一项外展计划，以提高公众的科学素养。该节目制作了一个简短的视频，解释三年级和四年级学生提出的科学问题，目的是展示科学如何以非常亲密的方式与他们的日常生活联系在一起。非蛋白质氨基酸L-精氨酸（L-End）和4，5-脱氢精氨酸（dhArg）分别是细菌产生的天然产物甘露肽霉素和吲哚霉素的成分。L-End和dhArg均由PLP依赖性氧化酶产生。来自和田山链霉菌（SwMppP）的酶使用分子氧在L-Arg底物侧链的碳4处安装羟基，而来自类芽孢杆菌（PeMppP）的酶在碳4和5之间产生双键（PeMppP）。SwMppP和PeMppP具有几乎相同的三级结构并且共享相同的活性位点残基。唯一的结构差异似乎是在二聚体界面，导致的假设，即在四级结构的差异影响的形状的活性位点，从而结合模式的L-精氨酸底物。一个主要的问题是这两种结构相似的酶如何通过不同的化学机制合成不同的产物。虽然关于SwMppP和PeMppP的机制已经知道很多（或可以推断），但这两种机制都没有完全表征。在这个项目中，新的替代底物3，4-脱氢-L-精氨酸被用来绕过早期阶段，从而通过停流和猝灭流动力学，NMR光谱和质谱实验探测SwMppP催化机制的后期阶段。类似的策略被用来研究PeMppP的机制。这项研究的结果将详细介绍这两种MppP样蛋白的反应机制，以及进化如何修改I型转氨酶折叠以执行新的催化功能。这些成果扩展了我们对PLP依赖性酶的具体知识以及对酶结构与功能关系的总体知识，并提高了蛋白质功能预测的准确性。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响进行评估，被认为值得支持。审查标准。