Investigating the oxidative chemistry and electron transfer in polysaccharide monooxygenases

项目摘要 多糖单加氧酶（PMO）也称为裂解性PMO（LPMO）是最近鉴定的一类多糖单加氧酶， 氧化降解多糖的酶。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。最近的兴趣已经转向了一个角色， 增强致病性。PMO存在于人类和植物病原体中。例如Magnaportha， 引起稻瘟病的生物体含有参与植物定殖的PMO。上调推定的 PMO也在人类感染粪肠球菌中发现，并且预测的PMO已经在人类感染粪肠球菌中发现。 粘质沙雷氏菌，炭疽杆菌，嗜肺军团菌。PMO作为毒力的新作用 这些因素表明，它们将成为对人类健康具有广泛影响的重要目标。理解 PMO的作用机制将为未来的研究和药物设计提供信息。PMO通过以下方式脱除纤维素： 在C1或C4碳上的氧化羟基化导致糖苷键的断裂。多糖 氧化通过PMO催化的O2的还原活化发生，其然后将O原子插入C1或C2中。 C4 C-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 提示功能。有三个高度保守的氨基酸区域。第一种称为组氨酸 在活性部位结合铜的支架。另外两个区域由Trp和Tyr链组成， 被推测为电子传输的管道。PMO的反应需要适时的 将多个电子传递到铜中心。纤维二糖脱氢酶（CDH）已被鉴定为一种 氧化还原与真菌PMO配对并由氧化纤维二糖的黄素结构域组成， 随后减少细胞色素结构域。细胞色素结构域是转移电子所必需的， 催化循环中的PMO。这里提出的研究试图回答四个主要问题： 在CDH和PMO之间转移，电子在CDH和PMO之间传递的时间性质是什么？ CDH和PMO，如何理解这个电子传递系统，使我们了解活性位点 机制，以及如何利用它来观察活性中间体？为了回答这些问题， 将表达和纯化CDHs和PMO，用于在有限电子轰击下的动力学和产物谱研究。 加载中通过诱变、蛋白质修饰，这些CDHs和PMO将包括新的性质， 将以可预测的方式扰动电子转移链， 转移蛋白质修饰将涉及基于Ru的光敏剂，其将允许对蛋白质的时间控制。 电子的传递。这些生化实验将与补充的 测量，如停流吸收光谱和高分辨率质谱。

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.