Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

Response of Brazilian peppertree (Schinus terebinthifolia) and four mangrove species to imazamox and carfentrazone-ethyl herbicides

Mangroves are a critical component of many coastal ecosystems in Florida. Woody species including Brazilian peppertree (Schinus terebinthifolia) have invaded thousands of hectares of mangrove habitat. The difficulty associated with ground-based management of invasive plants in mangrove communities has warranted a need to identify selective herbicides that can be applied aerially. Recent work suggests that Florida mangrove species are extremely sensitive to synthetic auxin herbicides; however, other herbicides have yet to be tested for selectivity. Greenhouse studies in 2018 and 2019 evaluated broadcast foliar applications of the acetolactate synthase (ALS) inhibitor imazamox and protoporphyrinogen oxidase (PPO) inhibitor carfentrazone-ethyl, both as individual treatments and in combinations, for control of S. terebinthifolia and injury to four non-target mangrove species. Across all post-treatment sample dates and species tested, there were no significant interactions between imazamox applied at 0.28 or 0.56 kg ai ha−1 in combination with carfentrazone-ethyl applied at 0 or 0.1 kg ha−1. Main effects of imazamox applied at 0.56 kg ai ha−1 and carfentrazone-ethyl applied at 0.1 kg ha−1 resulted in 99 and 97% defoliation, respectively, to Schinus terebinthifolia at 180 DAT. However, S. terebinthifolia % survival was 56 and 44% for the same treatments. Both herbicides severely injured all four mangroves by 90 DAT and resulted in 58 to 100% defoliation across species. At 180 DAT, significant increases in % cambium kill were also observed for all four species. Across species, mangrove survival varied but Rhizophora mangle survival was reduced to 6% when imazamox was applied at 0.56 kg ha−1. These results indicate both imazamox and carfentrazone-ethyl exhibit activity on S. terebinthifolia, but also injure all four mangroves enough to preclude their use as selective treatments.

Safety and efficacy of linuron with or without an adjuvant or S-metolachlor for POST control of Palmer amaranth (Amaranthus palmeri) in sweetpotato

Field studies were conducted to evaluate linuron for POST control of Palmer amaranth in sweetpotato to minimize reliance on protoporphyrinogen oxidase (PPO)-inhibiting herbicides. Treatments were arranged in a two by four factorial where the first factor consisted of two rates of linuron (420 and 700 g ai ha−1), and the second factor consisted of linuron applied alone or in combinations of linuron plus a nonionic surfactant (NIS) (0.5% v/v), linuron plus S-metolachlor (800 g ai ha−1), or linuron plus NIS plus S-metolachlor. In addition, S-metolachlor alone and nontreated weedy and weed-free checks were included for comparison. Treatments were applied to ‘Covington’ sweetpotato 8 d after transplanting (DAP). S-metolachlor alone provided poor Palmer amaranth control because emergence had occurred at applications. All treatments that included linuron resulted in at least 98 and 91% Palmer amaranth control 1 and 2 wk after treatment (WAT), respectively. Including NIS with linuron did not increase Palmer amaranth control compared to linuron alone, but increased sweetpotato injury and subsequently decreased total sweetpotato yield by 25%. Including S-metolachlor with linuron resulted in the greatest Palmer amaranth control 4 WAT, but increased crop foliar injury to 36% 1 WAT compared to 17% foliar injury from linuron alone. Marketable and total sweetpotato yield was similar between linuron alone and linuron plus S-metolachlor or S-metolachlor plus NIS treatments, though all treatments resulted in at least 39% less total yield than the weed-free check resulting from herbicide injury and/or Palmer amaranth competition. Because of the excellent POST Palmer amaranth control from linuron 1 WAT, a system including linuron applied 7 DAP followed by S-metolachlor applied 14 DAP could help to extend residual Palmer amaranth control further into the critical period of weed control while minimizing sweetpotato injury.

PPO-Inhibiting Herbicides and Structurally Relevant Schiff Bases: Evaluation of Inhibitory Activities against Human Protoporphyrinogen Oxidase

The study of human protoporphyrinogen oxidase (hPPO) inhibition can contribute significantly to a better understanding of some pathogeneses (e.g., porphyria, herbicide exposure) and the development of anticancer agents. Therefore, we prepared new potential inhibitors with Schiff base structural motifs (2-hydroxybenzaldehyde-based Schiff bases 9–13 and chromanone derivatives 17–19) as structurally relevant to PPO herbicides. The inhibitory activities (represented by the half maximal inhibitory concentration (IC50) values) and enzymatic interactions (represented by the hPPO melting temperatures) of these synthetic compounds and commercial PPO herbicides used against hPPO were studied by a protoporphyrin IX fluorescence assay. In the case of PPO herbicides, significant hPPO inhibition and changes in melting temperature were observed for oxyfluorten, oxadiazon, lactofen, butafenacil, saflufenacil, oxadiargyl, chlornitrofen, and especially fomesafen. Nevertheless, the prepared compounds did not display significant inhibitory activity or changes in the hPPO melting temperature. However, a designed model of hPPO inhibitors based on the determined IC50 values and a docking study (by using AutoDock) found important parts of the herbicide structural motif for hPPO inhibition. This model could be used to better predict PPO herbicidal toxicity and improve the design of synthetic inhibitors.

Evaluation of Preemergence Herbicide Programs for Control of Protoporphyrinogen Oxidase-Resistant Amaranthus palmeri in Soybean

Protoporphyrinogen oxidase- (PPO-) resistant Amaranthus palmeri (S.) Wats. (Palmer amaranth) was confirmed in Arkansas in 2015. Field trials were conducted in Crawfordsville, Gregory, and Marion, Arkansas in 2016, and Crawfordsville and Marion in 2017, assessing PPO-resistant Palmer amaranth control options in Glycine max (L.) Merr. (soybean). Twelve trials consisted of 26 preemergence (PRE) treatments, evaluated for Palmer amaranth control and density reduction at 28 days after treatment (DAT). Treatments that consisted of PPO- or acetolactate synthase- (ALS-) inhibiting herbicides such as flumioxazin (72 g ai ha−1) or sulfentrazone + cloransulam (195 g ha−1 + 25 g ha−1) controlled Palmer amaranth <60%. At 28 DAT, treatments including mixtures of a very-long-chain fatty acid (VLCFA) plus the photosystem II- (PSII-) inhibiting herbicide metribuzin provided increased control over single herbicide sites of action (SOA) or herbicides mixtures to which Palmer amaranth was resistant. Pyroxasulfone + metribuzin (149 g ha−1 + 314 g ha−1) controlled Palmer amaranth 91% control across twelve trials at 28 DAT. S-metolachlor alone did not provide consistent, acceptable control of PPO-resistant Palmer amaranth (55–77%); subsequent research has determined that these populations are resistant to S-metolachlor. A minimum of two effective herbicides should be included in soybean PRE programs for control of PPO-resistant Palmer amaranth.

DFT Based QSAR Studies of Phenyl Triazolinones of Protoporphyrinogen Oxidase Inhibitors

The quantitative structure activity relationships (QSARs) have been investigated on a series of substituted phenyl triazolinones having protoporphyrinogen oxidase (PPO) inhibition activities. The density functional theory (DFT) method is applied to calculate the quantum chemical descriptors. The derived QSAR model is based on three molecular descriptors namely highest occupied molecular orbital (HOMO) energy, electrophilic group frontier electron density (Fg E) and nucleus independent chemical shift (NICS). The best QSAR model has a square correlation coefficient r2 =0.886 and cross-validated square correlation coefficient q2 = 0.837.

Conformation of the Intermediates in the Reaction Catalyzed by Protoporphyrinogen Oxidase: An In Silico Analysis

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.

Influence of sulfentrazone and metribuzin applied preemergence on soybean development and yield

The use of photosystem II (PSII)-inhibitor and/or protoporphyrinogen oxidase (PPO)-inhibitor PRE herbicides in soybean may, under adverse environmental conditions, result in early season crop injury. A field study was conducted near Brule and North Platte, Nebraska, during the 2016 and 2017 growing seasons with the objective to evaluate the impact of PRE herbicides metribuzin (PSII-inhibitor) and sulfentrazone (PPO-inhibitor) on early season soybean development, final plant stand, and yield using 22 soybean varieties adapted to southwestern Nebraska. Herbicide treatments consisted of metribuzin (560 g ai ha−1) and sulfentrazone (280 g ai ha−1) applied within 3 d after planting and a nontreated control (NTC). Sulfentrazone reduced green canopy vegetation at the V2 growth stage by 22% and final plant stand at physiological maturity by 10% compared with the NTC. The number of pods per plant was 16% higher for sulfentrazone and the number of seeds per plant was 15% and 4% higher for sulfentrazone and metribuzin compared with the NTC, respectively. Sulfentrazone and metribuzin resulted in a slightly higher yield (3%) compared with the NTC, thus no yield reduction from PRE herbicides was observed in this study. These results support other findings that sulfentrazone and metribuzin have potential to cause early-season crop injury; however, when applied according to their label recommendations and following regional agronomic management practices, this impact may not translate into soybean yield reduction while such herbicides provide effective soil residual weed control.