Friday, April 19, 2024
4/19/2024
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.
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