Investigating the oxidative chemistry and electron transfer in polysaccharide monooxygenases

研究多糖单加氧酶的氧化化学和电子转移

• 批准号: 10611373
• 负责人: Richard Sayler
• 金额: $ 6.97万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10611373
• 关键词: Active Sites; Affect; Agriculture; Amines; Amino Acids; Antibiotic Resistance; Bacillus anthracis; Binding; Biochemical; Biomass; Buffers; Carbon; Catalysis; Cellulose; Chemistry; Chromatography; Collaborations; Consumption; Copper; Cysteine; Cytochrome a; Cytochromes; Deuterium; Drug Design; Educational process of instructing; Electron Transport; Electrons; Enterococcus faecalis; Enzymes; Family; Flavins; Future; Gallic acid; Health; Histidine; Human; Hydrogen Bonding; Hydroxylation; Imidazole; In Vitro; Infection; Ions; Isotope Labeling; Isotopes; Kinetics; Legionella pneumophila; Libraries; Lytic; Mass Spectrum Analysis; Measurement; Measures; Methanol; Mixed Function Oxygenases; Molecular; Mutagenesis; Mutate; N-terminal; Nature; Ohio; Optics; Organism; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygenases; Pathogenicity; Performance; Photosensitizing Agents; Physiological; Pichia; Plants; Play; Polysaccharides; Post-Translational Protein Processing; Property; Proteins; Protons; Reaction; Recombinants; Reducing Agents; Research; Research Design; Resolution; Rice; Role; Serratia marcescens; Solvents; Spectrum Analysis; Surface; System; Time; Up-Regulation; Virulence Factors; absorption; ascorbate; depolymerization; emission spectroscopy; experimental study; expression vector; inducible gene expression; insight; interest; mutant; oxidation; pathogen; pressure; small molecule; success

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.

项目摘要 多糖单加氧酶（PMO）也称为裂解PMO（LPMO）是最近确定的一类 氧化降解多糖的酶。对PMO的兴趣主要集中在利用 他们对植物生物量降解产生生物燃料的作用。最近的兴趣转向了 增强致病性。 PMO在人类和植物病原体中发现。例如Magnaportha oryzae， 引起大米爆炸的生物包含参与植物定植的PMO。推定的上调 在人类感染粪肠球菌中也发现了PMO，并且在 Serratia marcescens，炭疽芽孢杆菌和肺炎军团菌。 PMO作为毒力的新兴作用 因素表明，它们将成为重要的目标，对人类健康有广泛的影响。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解聚纤维素通过 C1或C4碳的氧化羟基化导致糖苷键的切割。多糖 氧化是通过PMO催化的O2的还原性激活发生的，然后将O-ATOM插入C1或 C4 C-H键。所有PMO都被认为具有共同的机制，因此保守的活动现场残留物提供 提示功能。高度保守的氨基酸有三个区域。第一个被称为组氨酸 支撑在活性位点结合铜。其他两个区域由TRP和Tyr链组成 被推测是电子传输的导管。 PMO反应需要及时的 将多个电子传递到铜中心。纤维二酶脱氢酶（CDH）已被鉴定为 氧化还原与真菌PMO的合作伙伴，由黄素结构域组成，该结构域氧化了纤维二糖，该结构粒氧化 随后减少了细胞色素域。将电子转移到 催化周期中的PMO。这里提出的研究试图回答四个主要问题：电子如何 转移在CDH和PMO之间，电子之间的递送时间是什么 CDH和PMO，对该电子输送系统的理解如何告知我们活动地点 机制，如何利用观察反应性中间体？要回答这些问题， 在有限的电子下，CDH和PMO将在动力学和产品概况研究中表达和纯化 加载中。通过诱变，蛋白质修饰，这些CDH和PMO将包括新特性 将以可预测的方式扰动电子传输链，从而提供有关电子的分子信息 转移。蛋白质修饰将涉及基于RU的光敏剂，该光敏剂将允许对 电子的传递。这些生化实验将与互补一起进行 测量值，例如停止吸光度光谱和高分辨率质谱。