benzofurans and isoliquiritigenin

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

Topics: Amino Acid Sequence; Benzofurans; Binding Sites; Catalytic Domain; Catechol Oxidase; Chalcones; Copper; Coreopsis; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Plant Proteins; Protein Conformation; Substrate Specificity; Tyramine

It is becoming increasingly important to investigate drug metabolites to evaluate their toxic or preventive effects after administration of the parent compound. In our previous study, isoliquiritigenin isolated from Glycyrrhizae Radix effectively protected mouse-derived hippocampal neuronal cells (HT22) against 5mM glutamate-induced oxidative stress. However, there is little information on the protective effects of the metabolites of isoliquiritigenin on HT22 cells. In this study, isoliquiritigenin and its Phase I metabolites were prepared and their neuroprotective activities on glutamate-treated HT22 cells were compared. The prepared metabolites were liquiritigenin (1), 2',4,4',5'-tetrahydroxychalcone (2), sulfuretin (3), butein (4), davidigenin (5), and cis-6,4'-dihydroxyaurone (6). Among the six metabolites, 4 showed better neuroprotective effects than the parent compound, isoliquiritigenin. Our study suggests that the neuroprotective effect of isoliquiritigenin could be elevated by its active metabolite 4, which is a chalcone containing a catechol group in the B ring.

Topics: Animals; Benzofurans; Cell Death; Cell Line; Chalcone; Chalcones; Flavonoids; Glutamic Acid; Hippocampus; Mice; Neurons; Neuroprotective Agents

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation.

Topics: Amino Acid Sequence; Benzofurans; Cells, Cultured; Chalcones; Chromatography, High Pressure Liquid; Cloning, Molecular; Gene Expression Profiling; Glucosides; Medicago truncatula; Metabolomics; Molecular Sequence Data; Molecular Structure; Oligonucleotide Array Sequence Analysis; Peroxidases; Recombinant Proteins; RNA, Plant; Substrate Specificity; Triterpenes

To study the chemical constituents from the root of Millettia speciosa.. To isolate and purify by silica gel, macroporous resin D-101 and Sephadex LH-20.. Five compounds were isolated from 95% EtOH extract of Millettia Speciosa and their structures were elucidated by physico-chemical properties and spectroscopic analysis as Isoliquiritigenin (I), Maackiain (II), Pterocarpin (III), Medicarpin (IV) and Homopterocarpin (V).. Compounds I, III are obtained from this plant for the first time, compounds V is isolated from the genus Millettia for the first time.

Topics: Benzofurans; Benzopyrans; Chalcones; Ethanol; Magnetic Resonance Spectroscopy; Millettia; Molecular Structure; Plant Roots; Plants, Medicinal; Pterocarpans