Abstract:

Currently, 2.37 billion individuals cannot acquire enough food, and such food insecurity is only worsened by fungal pathogens. For instance, penicillium digitatum (Pd) accounts for 90% of all post-harvest citrus crop loss, threatening a critical portion of the world's food supply. Cdc14 enzymes, which dephosphorylate cyclin-dependent kinases, have shown promise in inhibiting fungal growth in species like S. cerevisiae and F. graminearum, as they prevent cellular replication. Due to this enzyme's critical role in the cell cycle, it functions as a powerful target for constraining Pd. This investigation began by characterizing the Cdc14 enzyme in Pd, using E. Coli cells to generate the protein, and then conducting experiments to ensure the enzyme was purified correctly. SDS-PAGE confirmed proper purification and ascertained the protein molecular weight to be 53.2 kDa, while the pNPP assay verified the phosphatase nature of the protein. Kinetic assay results yielded a Km of 10 ± 5uM and a kcat at 1.1±0.2 seconds-1, and a specificity assay showed greater affinity occurred in phosphopeptide substrates with a phosphorylated serine, a +1 proline, a +3 lysine, and another lysine or arginine. Following enzyme characterization, inhibition experimentation began, where aurintricarboxylic acid (I1) was found to have the best inhibition properties in vitro and in silico, with 58.538% inhibition and a -6.1815 binding score, respectively. The IC50 value for I1 was then calculated at 18.6 uM before the inhibitors were optimized in silico to create a novel fungicide, which opens up numerous future research options.