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Browsing Theses and Dissertations by Author "Barker, Abigail, author"

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    Protoporphyrinogen oxidase: origins, functions, and importance as an herbicide target site
    (Colorado State University. Libraries, 2021) Barker, Abigail, author; Dayan, Franck, advisor; Snow, Christopher, committee member; Pilon, Marinus, committee member; Gaines, Todd, committee member
    Protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides are effective tools to control a broad spectrum of weeds, including those that have evolved resistance to glyphosate. Their utility is being threatened by the appearance of biotypes that are resistant to PPO inhibitors. While the chloroplastic PPO1 isoform is thought to be the primary target of PPO herbicides, evolved resistance mechanisms elucidated to date are associated with changes to the mitochondrial PPO2 isoform, suggesting that the importance of PPO2 has been underestimated. Our investigation of the evolutionary and structural biology of plant PPOs provides some insight into the potential reasons why PPO2 is the preferred target for evolution of resistance. The most common target-site mutation imparting resistance involved the deletion of a key glycine codon. The genetic environment that facilitates this deletion is apparently only present in the gene encoding PPO2 in a few species. Additionally, both species with this mutation (Amaranthus tuberculatus and Amaranthus palmeri) have dual targeting of PPO2 to both the chloroplast and the mitochondria, which might be a prerequisite to impart herbicide resistance. The most recent target-site mutations have substituted a key arginine residue involved in stabilizing the substrate in the catalytic domain of PPO2. This arginine is highly conserved across all plant PPOs, suggesting that its substitution could be equally likely on PPO1 and PPO2, yet it has only occurred on PPO2, underscoring the importance of this isoform for the evolution of herbicide resistance. As glyphosate resistance becomes widespread, weed control turns to older mechanisms of action with less resistance. Protoporphyrinogen oxidase (PPO) inhibitors are a versatile class of herbicides that have been used since the 1960's, with active ingredients that work in pre-emergent and post-emergent applications. Differential efficacy of PPO inhibitors applied pre-emergent, early post-emergent and late post-emergent has been observed in multiple species and settings. Understanding the cause of higher efficacy in younger plants could preserve these important weed control tools. To understand the differing efficacies elements that affect the mechanism of action of PPO inhibitors were analyzed over the course of plant growth including target site transcript levels and protein levels, herbicide uptake, antioxidant capacity, and indicators of flux through the pathway. Data show levels of PPO do not explain differential efficacy. Increases of glutamate, the pathway precursor, do increase damage due to PPO inhibitor treatment, but increased levels are not observed in younger plants. Differential efficacy is likely due to a combination of increase in antioxidant capacity and a decrease in herbicide uptake. Other possible factors such as metabolism will need to be measured in future work. Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204–210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the β-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction. Weed control is essential in modern agriculture, though it becomes more difficult with increasing resistance levels to current herbicides and a slow process to register a new mechanisms of action because of safety concerns and current methods. Agrematch provides a new method to identify possible herbicide candidates using an artificial intelligence algorithm that takes into effect biological parameters with the goal of reducing R&D time on new herbicides. Herein we describe the discovery of 4-chloro-2-pentenamides as novel inhibitors of protoporphyrinogen oxidase, a known herbicide target site, by the Agrematch AI. The herbicidal activity is confirmed in greenhouse assays, with the highest performing AGR001 showing good activity pre-emergent at 150 g/ha and post emergent as low as 50 g/ha on the troublesome weed palmer amaranth (Amaranthus palmeri). A lack of activity is shown on PPO resistant palmer amaranth carrying the ΔG210 deletion mutation. The mechanism of action is confirmed by the herbicide dependent accumulation of protoporphyrin IX, subsequent light dependent loss of membrane integrity, and direct inhibition of protoporphyrinogen oxidase in an in vitro assay. Modeling of the docking of these inhibitors in the active site of protoporphyrinogen oxidase confirms the target.