Identification of a Novel 2,4-D Metabolic Detoxification Pathway in 2,4-D-Resistant Waterhemp (Amaranthus tuberculatus).

A 2,4-dichlorophenoxyactic acid (2,4-D)-resistant population of Amaranthus tuberculatus (common waterhemp) from Nebraska, USA, was previously found to have rapid metabolic detoxification of the synthetic auxin herbicide 2,4-D. We purified the main 2,4-D metabolites from resistant and susceptible plants, solved their structures by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and synthesized the metabolites to determine their in planta toxicity. Susceptible plants conjugated 2,4-D to aspartate to form 2,4-D-aspartic acid (2,4-D-Asp), while resistant plants had a unique metabolic profile where 2,4-D was hydroxylated into 5-OH-2,4-D, followed by conjugation into a sugar metabolite (2,4-D-5-O-d-glucopyranoside) and subsequent malonylation into 2,4-D-(6'-O-malonyl)-5-O-d-glucopyranoside. Toxicological studies on waterhemp and Arabidopsis thaliana confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite found in susceptible plants retained some auxinic action and toxicity. These results demonstrate that 2,4-D-resistant A. tuberculatus evolved novel detoxification reactions not present in susceptible plants to rapidly metabolize 2,4-D, potentially mediated by cytochrome P450 enzymes that perform the initial 5-hydroxylation reaction. This novel mechanism is more efficient to detoxify 2,4-D and produces metabolites with lower toxicity compared to the aspartic acid conjugation found in susceptible waterhemp.

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.