Structural determinants of reaction specificity in PLP-dependent arginine oxidases

生物化学中的一般理解是，酶的分子结构决定其功能，因此，具有相同或非常相似结构(同系物)的两种酶应催化相同或相似的化学反应。通过这一奖项，化学部的生命过程化学项目资助了威斯康星大学密尔沃基大学的Nicholas Silvaggi博士，以确定来自两个不同生物体的酶的同系物如何使用不同的化学步骤产生不同的产品。沃达雅链霉菌的5‘-磷酸吡哆醛-5’-磷酸L精氨酸脱氨酶是由西尔瓦吉博士发现的，它能与分子氧反应，在氨基酸精氨酸上加一个羟基。来自埃尔吉拟杆菌的酶(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。这两种产品最终都会被合并到包括抗生素在内的次生代谢物中。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，并更好地理解现有的酶结构如何适应执行新的功能。西尔瓦吉博士制定了一项外展计划，以提高公众的科学素养。该节目制作了三年级和四年级学生提出的科学问题的短片讲解，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-内尿酸(L-完)和4，5-脱氢精氨酸(DhArg)分别是细菌产生的天然产品甘露糖霉素和吲哚霉素的成分。L末端和dhArg都是由PLP依赖的氧化酶产生的。瓦达雅链霉菌的酶(SwMppP)利用分子氧在L-精氨酸底物侧链的碳4上安装羟基，而埃尔基拟杆菌(PeMppP)的酶(PeMppP)在碳4和碳5(PeMppP)之间产生双键。SwMppP和PeMppP具有几乎相同的三级结构和相同的活性中心残基。唯一的结构差异似乎是在二聚体界面，这导致了四级结构的差异影响活性中心的形状，从而影响L-精氨酸底物的结合模式。正在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品。虽然关于SwMppP和PeMppP的机制已经知道(或可以推断)很多，但这两种机制都没有完全表征。本项目采用新型底物3，4-脱氢-L-精氨酸作为替代底物，通过停流和急流动力学、核磁共振谱和质谱学实验，对SWMppP催化反应的早期阶段和后期阶段进行了研究。类似的策略也被用来研究PeMppP的机制。这项研究的结果将是关于这两个MPPP样蛋白的反应机制的详细信息，以及因此进化如何修改I型转氨酶折叠来执行新的催化功能。这些结果扩展了我们对PLP依赖的酶的专门知识和一般酶结构-功能关系的知识，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智力优势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.