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 ß-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.

Fate of Glyphosate during Production and Processing of Glyphosate-Resistant Sugar Beet ( Beta vulgaris).

Glyphosate is a widely used herbicide in commercial crop production for both conventional and herbicide-resistant crops. Herbicide-resistant crops, like glyphosate-resistant sugar beet, are often exposed to multiple applications of glyphosate during the growing season. The fate of this herbicide in resistant crops has not been publicly documented. We investigated the fate of glyphosate and main metabolite aminomethylphosphonic acid in glyphosate-resistant sugar beet grown in northern Colorado. Glyphosate residues were measured via directed ultra-high-performance liquid chromatography tandem mass spectrometry analysis of sugar beet shoots and roots throughout the growing season, from samples collected at various steps during sugar beet processing, and from flow-through samples of greenhouse-grown beets. Sugar beet rapidly absorbed glyphosate after foliar application, and subsequently translocated the herbicide to its roots, with between 2 and 3 µg/g fresh weight measured in both tissue types within 1 week of application. However, only trace amounts of glyphosate remained in either the shoots or the roots 2 weeks after application. Analysis of irrigation flow-through in pot assays confirmed that the herbicide readily exuded out of the roots. Processing of the beets removed glyphosate and herbicide levels were below the limit of detection in the crystalline sugar final product.

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.