Structural determinants of reaction specificity in PLP-dependent arginine oxidases

生物化学的一般理解是，酶的分子结构决定了它的功能，因此，具有相同或非常相似结构（同源物）的两种酶应该催化相同或相似的化学反应。有了这个奖项，化学部门的生命过程化学项目资助了威斯康星大学密尔沃基分校的尼古拉斯·西尔瓦吉博士，以确定来自两种不同生物体的酶的同源物如何通过不同的化学步骤产生不同的产品。来自wadayamstreptomyces wadayamensis （SwMppP）的pyridoxal-5'-phosphate-dependent L-arginine deaminase（或MppP）酶被Silvaggi博士发现可以与分子氧反应，在氨基酸精氨酸上添加一个羟基。来自elgii Paenibacillus （PeMppP）的酶具有与SwMppP几乎相同的结构，但在精氨酸侧链的中间产生一个新的双键。这两种产品最终被纳入次级代谢物，包括抗生素。从这些研究中获得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执行新功能。西尔瓦吉博士制定了一项推广计划，以提高公众的科学素养。这个节目制作了三年级和四年级学生提出的科学问题的简短视频解释，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。非蛋白氨基酸L-enduracididine （L-End）和4,5-dehydroarginine （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都是由plp依赖性氧化酶产生的。来自wadayamstreptomyces wadayamensis （SwMppP）的酶利用分子氧在L-Arg底物侧链的碳4上安装羟基，而来自elgii Paenibacillus （PeMppP）的酶在碳4和碳5之间创建双键（PeMppP）。SwMppP和PeMppP具有几乎相同的三级结构和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上，这导致了四元结构的差异影响活性位点形状的假设，从而影响了L-Arg底物的结合模式。要解决的一个主要问题是，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。虽然我们对SwMppP和PeMppP的机制了解很多（或可以推断），但这两种机制都没有得到充分的表征。本项目利用新型替代底物3,4-脱氢- l -精氨酸绕过早期阶段，通过停流和淬流动力学、核磁共振波谱和质谱实验探索SwMppP的后期催化机制。采用类似的策略来研究PeMppP的机制。本研究的结果将详细说明这两种mppp样蛋白的反应机制，以及进化如何改变I型转氨酶折叠以发挥新的催化功能。这些结果扩展了我们对plp依赖性酶的知识，特别是酶的结构-功能关系，并提高了蛋白质功能预测的准确性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.