Identification and Characterization of Glycosyltransferases Involved in the Biosynthesis of Neodiosmin.

Glycosylation plays a very important role in plant secondary metabolic modifications. Neodiosmin, identified as diosmetin-7-O-neohesperidoside, not only acts to mitigate bitterness and enhance the flavor of food but also serves as a pivotal metabolite that reinforces plant immunity. Investigating its biosynthetic pathway in plants is crucial for optimizing fruit quality and fortifying plant immune responses. In this study, through analysis of transcriptomic data from Astilbe chinensis, we identified two novel uridine diphosphate (UDP)-glycosyltransferases (UGTs): Ach14791 (AcUGT73C18), responsible for flavonoid 7-O-glycosylation and Ach15849 (AcUGT79B37), involved in flavonoid-7-O-glucoside-2″-O-rhamnosylation. By delving into enzymatic properties and catalytic promiscuity, we developed a biosynthesis route of neodiosmin by establishing a one-pot enzyme-catalyzed cascade reaction. Simultaneously, lonicerin and rhoifolin were also successfully synthesized using the same one-pot dual-enzyme catalytic reaction. Taken together, our findings not only identified two novel UGTs involved in neodiosmin biosynthesis but also provided important biocatalytic components for the microorganism-based biosynthesis of flavonoid-7-O-disaccharide compounds.

Combination of 2-tert-Butyl-1,4-Benzoquinone (TBQ) and ZnO Nanoparticles, a New Strategy To Inhibit Biofilm Formation and Virulence Factors of Chromobacterium violaceum.

Drug-resistant bacteria have been raising serious social problems. Bacterial biofilms and different virulence factors are the main reasons for persistent infections. As a conditioned pathogen, Chromobacterium violaceum has evolved a vast network of regulatory mechanisms to modify and fine-tune biofilm development, contributing to multidrug resistance. However, there are few therapies to combat drug-resistant bacteria. Quorum sensing (QS) inhibitors (QSIs) are a promising strategy to solve antibiotic resistance. Our previous work suggested that 2-tert-butyl-1,4-benzoquinone (TBQ) is a potent QSI. In this study, the combination of zinc oxide nanoparticles (ZnO-NPs) and TBQ (ZnO-TBQ) was investigated for the treatment of Chromobacterium violaceum ATCC 12472 infection. ZnO-NPs attach to cell walls or biofilms, and the local dissolution of ZnO-NPs can lead to increased Zn2+ concentrations, which could destroy metal homeostasis, corresponding to disturbances in amino acid metabolism and nucleic acid metabolism. ZnO-NPs significantly improved the efficiency of TBQ in inhibiting the QS-related virulence factors and biofilm formation of C. violaceum ATCC 12472. ZnO-TBQ effectively reduces the expression of genes related to QS, which is conducive to limiting the infectivity of C. violaceum ATCC 12472. Caenorhabditis elegans nematodes treated with ZnO-TBQ presented a significant improvement in the survival rate by 46.7%. Overall, the combination of ZnO-NPs and TBQ offers a new strategy to attenuate virulence factors and biofilm formation synergistically in some drug-resistant bacteria. IMPORTANCE The combination of ZnO-NPs and TBQ (ZnO-TBQ) can compete with the inducer N-decanoyl-homoserine lactone (C10-HSL) by binding to CviR and downregulate genes related to the CviI/CviR system to interrupt the QS system of C. violaceum ATCC 12472. The downstream genes responding to cviR were also downregulated so that virulence factors and biofilm formation were inhibited. Furthermore, ZnO-TBQ presents multiple metabolic disturbances in C. violaceum ATCC 12472, which results in the reduced multidrug resistance and pathogenicity of C. violaceum ATCC 12472. In an in vivo assay, C. elegans nematodes treated with ZnO-TBQ presented a significant improvement in the survival rate by 46.7% by limiting the infectivity of C. violaceum ATCC 12472. In addition, ZnO-TBQ inhibited the generation of virulence factors and biofilm formation 2-fold compared to either ZnO-NPs or TBQ alone. The combination of ZnO-NPs with TBQ offers a potent synergistic strategy to reduce multidrug resistance and pathogenicity.

Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity.

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N-methyltransferase (LcNMT1) and two O-methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)-coclaurine to produce mono- and dimethylated products. Mutagenesis experiments revealed that a single-residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)-norcoclaurine at the C6 site and LcOMT3 methylates (S)-coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales-derived NMTs, Magnoliales-derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.

Design, synthesis, and biological evaluation of quinazolin-4(3H)-one derivatives co-targeting poly(ADP-ribose) polymerase-1 and bromodomain containing protein 4 for breast cancer therapy.

This study was aimed to design the first dual-target small-molecule inhibitor co-targeting poly (ADP-ribose) polymerase-1 (PARP1) and bromodomain containing protein 4 (BRD4), which had important cross relation in the global network of breast cancer, reflecting the synthetic lethal effect. A series of new BRD4 and PARP1 dual-target inhibitors were discovered and synthesized by fragment-based combinatorial screening and activity assays that together led to the chemical optimization. Among these compounds, 19d was selected and exhibited micromole enzymatic potencies against BRD4 and PARP1, respectively. Compound 19d was further shown to efficiently modulate the expression of BRD4 and PARP1. Subsequently, compound 19d was found to induce breast cancer cell apoptosis and stimulate cell cycle arrest at G1 phase. Following pharmacokinetic studies, compound 19d showed its antitumor activity in breast cancer susceptibility gene 1/2 (BRCA1/2) wild-type MDA-MB-468 and MCF-7 xenograft models without apparent toxicity and loss of body weight. These results together demonstrated that a highly potent dual-targeted inhibitor was successfully synthesized and indicated that co-targeting of BRD4 and PARP1 based on the concept of synthetic lethality would be a promising therapeutic strategy for breast cancer.