15 research outputs found
Recommended from our members
Polysaccharide Monooxygenases Involved in Non-Catabolic Physiological Processes
Polysaccharide Monooxygenases (PMOs) activate molecular oxygen through a copper active site to hydroxylate and subsequently cleave polysaccharides. These enzymes are of interest for their potential role in biofuel production and C-H activation chemistry. The biological role of many of the first described members of this superfamily family was in polysaccharide breakdown for carbon uptake carried out by fungi and bacteria. More recently, roles for these enzymes outside of carbon utilization have emerged. This dissertation will focus on two fungal PMOs and one bacterial PMO that fulfill roles other than catabolism. Chapter 1 outlines the current understanding of PMOs as a superfamily of enzymes. This introduction details the copper active site, known substrates of PMOs, and what is known about the steps involved in catalysis. The following three chapters focus on putative PMOs that have unique biological roles other than nutrient degradation for uptake by the organism. Chapter 2 focuses on the biochemical characterization of the PMO, CWR-1, and how it plays a role in the allorecognition process of Neurospora crassa. Evidence shows that CWR-1 enzymatic activity is not crucial for this role but is likely a PPI. Chapter 3 focuses on the putative PMO, HAM-7. This PMO is involved in fungal development, and N. crassa strains that lack this protein have severe growth phenotypes. The chapter details efforts to express and purify this protein to obtain more detailed biochemical information. Chapter 4 focuses on the Vibrio cholerae PMO, GBPA, and the proteases VcLAP and VcLAPX. GBPA has been shown to play a role in adherence to mucin during the infection lifecycle of the bacteria Vibrio cholerae. The potential activity of GBPA on mucin and the characterization of these proteases is presented
A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in <i>Neurospora crassa</i> allorecognition.
A dual-fluorophore sensor approach for ratiometric fluorescence imaging of potassium in living cells.
Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K+ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K+ physiology and pathology owing to the large differences in intracellular versus extracellular K+ concentrations ([K+]intra = 150 mM, [K+]extra = 3-5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K+ in biological specimens that meet the dual challenges of low affinity and high selectivity for K+, particularly over Na+, currently available fluorescent K+ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K+ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K+ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K+. RPS-1 responds to K+ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K+ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K+ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs
Recommended from our members
Characterization of a unique polysaccharide monooxygenase from the plant pathogen Magnaporthe oryzae.
Blast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase (MoPMO9A) is increased. MoPMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the MoPMO9A family AA9 showed that 220 of the 223 sequences in the MoPMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two MoPMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), MoPMO9A is not active on cellulose but showed activity on cereal-derived mixed (1→3, 1→4)-β-D-glucans (MBG). Moreover, the DUF is required for activity. MoPMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. MoPMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for MoPMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of MoPMO9A results in reduced pathogenicity
Abiotic sugar synthesis from CO2 electrolysis
CO2 valorization is aimed at converting waste CO2 to value-added products. While steady progress has been achieved through diverse catalytic strategies, including CO2 electrosynthesis, CO2 thermocatalysis, and biological CO2 fixation, each of these approaches have distinct limitations. Inorganic catalysts only enable synthesis beyond C2 and C3 products with poor selectivity and with a high energy requirement. Meanwhile, although biological organisms can selectively produce complex products from CO2, their slow autotrophic metabolism limits their industrial feasibility. Here, we present an abiotic approach leveraging electrochemical and thermochemical catalysis to complete the conversion of CO2 to life-sustaining carbohydrate sugars akin to photosynthesis. CO2 was electrochemically converted to glycolaldehyde and formaldehyde using copper nanoparticles and boron-doped diamond cathodes, respectively. CO2-derived glycolaldehyde then served as the key autocatalyst for the formose reaction, where glycolaldehyde and formaldehyde combined in the presence of an alkaline earth metal catalyst to form a variety of C4 - C8 sugars, including glucose. In turn, these sugars were used as a feedstock for fast-growing and genetically modifiable Escherichia coli. Altogether, we have assembled a platform that pushes the boundaries of product complexity achievable from CO2 conversion while demonstrating CO2 integration into life-sustaining sugars
Recommended from our members
A Prototype Assay Multiplexing SARS-CoV-2 3CL-Protease and Angiotensin-Converting Enzyme 2 for Saliva-Based Diagnostics in COVID-19.
With the current state of COVID-19 changing from a pandemic to being more endemic, the priorities of diagnostics will likely vary from rapid detection to stratification for the treatment of the most vulnerable patients. Such patient stratification can be facilitated using multiple markers, including SARS-CoV-2-specific viral enzymes, like the 3CL protease, and viral-life-cycle-associated host proteins, such as ACE2. To enable future explorations, we have developed a fluorescent and Raman spectroscopic SARS-CoV-2 3CL protease assay that can be run sequentially with a fluorescent ACE2 activity measurement within the same sample. Our prototype assay functions well in saliva, enabling non-invasive sampling. ACE2 and 3CL protease activity can be run with minimal sample volumes in 30 min. To test the prototype, a small initial cohort of eight clinical samples was used to check if the assay could differentiate COVID-19-positive and -negative samples. Though these small clinical cohort samples did not reach statistical significance, results trended as expected. The high sensitivity of the assay also allowed the detection of a low-activity 3CL protease mutant
A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition.
Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling), encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain
Recommended from our members
A Modular Ionophore Platform for Liver-Directed Copper Supplementation in Cells and Animals.
Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely on generic copper supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. Here we present a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, we designed and synthesized an N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying "Trojan Horse" that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease