Investigating the oxidative chemistry and electron transfer in polysaccharide monooxygenases

项目总结 多糖单加氧酶(PMOS)也被称为裂解PMOS(LPMOS)，是最近发现的一类 氧化降解多糖的酶。人们对PMO的兴趣主要集中在利用 它们对植物生物质降解产生生物燃料的作用。最近的兴趣转向了在 增强致病性。PMOS存在于人类和植物的病原体中。例如稻米木兰， 引起稻瘟病的有机体含有一种与植物定殖有关的PMO。推定的上调 在人类感染粪肠球菌中也发现了PMOS，预测的PMOS在 粘质沙雷氏菌、炭疽芽孢杆菌和嗜肺军团菌。PMO作为毒力的新角色 各种因素表明，它们将成为对人类健康具有广泛影响的重要目标。理解 PMOS的作用机制将为未来的研究和药物设计提供信息。PMOS通过解聚纤维素 在C1或C4碳上的氧化羟化导致糖苷键的断裂。多糖类 氧化是通过PMO催化的O2的还原活化发生的，O2然后将O-原子插入到C1或 C4C-H键。所有PMO被认为共享一个共同的机制，因此保守的活性位点残基提供了 关于功能的提示。有三个高度保守的氨基酸区域。第一种被称为组氨酸 将铜结合在活性部位的支撑。另外两个区域由Trp和Tyr链组成，它们 被认为是电子传输的管道。PMO的反应需要适时的 将多个电子传送到铜中心。纤维二糖脱氢酶(CDH)是一种 氧化还原与真菌PMO配对，由黄素区组成，黄素区氧化纤维二糖， 随后减少细胞色素结构域。细胞色素结构域是将电子转移到 PMOS在催化循环中。这里提出的研究试图回答四个主要问题：电子是如何 在CDH和PMO之间传输，电子在CDH和PMO之间传输的时间性质是什么 CDH和PMO，对这个电子传递系统的了解如何才能告诉我们活性部位 机制，以及如何利用它来观察活性中间体？要回答这些问题， CDHS和PMOS将被表达和纯化，用于有限电子条件下的动力学和产物谱研究 正在装车。通过突变、蛋白质修饰，这些CDHS和PMO将包括新的特性 将以可预测的方式扰乱电子转移链，提供有关电子的分子信息 调职。蛋白质修饰将涉及一种基于Ru的光敏剂，它将允许时间控制 电子的传递。这些生化实验将与补充 停流吸收光谱和高分辨率质谱仪等测量方法。

Project summary Polysaccharide monooxygenases (PMOs) also known as lytic PMOs (LPMOs) are a recently identified class of enzymes that oxidatively degrade polysaccharides. Interest in PMOs has largely been focused on harnessing their action for plant biomass degradation to generate biofuels. Recent interest has turned to a role in enhancing pathogenicity. PMOs are found in human and plant pathogens. For example Magnaportha oryzae, the organism that causes rice blast, contains a PMO involved in plant colonization. Upregulation of putative PMOs is also found in the human infection Enterococcus faecalis, and predicted PMOs have been found in Serratia marcescens, Bacillus anthracis, and Legionella pneumophila. The emerging role of PMOs as virulence factors suggest that they will be an important target with broad implication in human health. Understanding PMOs mechanism of action will inform future studies and drug design. PMOs depolymerize cellulose through oxidative hydroxylation at the C1 or C4 carbon leading to cleavage of the glycosidic bond. Polysaccharide oxidation occurs through PMO-catalyzed reductive activation of O2, which then inserts a O-atom into a C1 or C4 C-H bond. All PMOs are thought to share a common mechanism, thus conserved active site residues offer hints as to function. There are three regions of highly conserved amino acids. The first is termed the histidine brace which binds copper in the active site. The two other regions are composed of Trp and Tyr chains that have been speculated to serve as conduits for electron transport. The PMO reaction requires the well-timed delivery of multiple electrons to the copper center. Cellobiose dehydrogenase (CDH) has been identified as a redox partners with fungal PMOs and is composed of a flavin domain that oxidizes cellobiose which, subsequently reduces a cytochrome domain. The cytochrome domain is required for the transfer electrons to PMOs in the catalytic cycle. The studies proposed here seek to answer four main questions: How are electrons transferred between the CDH and the PMO, what is the temporal nature of the delivery of electrons between the CDH and the PMO, how can the understanding of this electron delivery system inform us of the active site mechanism, and how can it be harnessed to observe reactive intermediates? To answer these questions, CDHs and PMOs will be expressed and purified for kinetic and product profile studies under limited electron loading. Through mutagenesis, protein modification, these CDHs and PMOs will include new properties that will perturb the electron transfer chain in a predictable manner providing molecular information on electron transfer. The protein modification will involve a Ru based photosensitizer that will allow temporal control over the delivery of electrons. These biochemical experiments will be performed in conjunction with complementary measurements such as stop-flow absorbance spectroscopy and high-resolution mass spectrometry.