Search Results (8)

Hairy vetch (Vicia villosa Roth) and smooth vetch (V. villosa Roth var. glabrescens) are important cover crops and legume forage with great economic and ecological values. Due to the large and highly heterozygous genome, full-length transcriptome reconstruction is a cost-effective route to mining their genetic resources. In this study, a hybrid sequencing approach combining SMRT and NGS technologies was applied. The results showed that 28,747 and 40,600 high-quality non-redundant transcripts with an average length of 1808 bp and 1768 bp were generated from hairy vetch and smooth vetch, including 24,864 and 35,035 open reading frames (ORFs), respectively. More than 96% of transcripts were annotated to the public databases, and around 25% of isoforms underwent alternative splicing (AS) events. In addition, 987 and 1587 high-confidence lncRNAs were identified in two vetches. Interestingly, smooth vetch contains more specific transcripts and orthologous clusters than hairy vetch, revealing intraspecific transcript diversity. The phylogeny revealed that they were clustered together and closely related to the genus Pisum. Furthermore, the estimation of Ka/Ks ratios showed that purifying selection was the predominant force. A putative 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SDH) gene underwent strong positive selection and might regulate phenotypic differences between hairy vetch and smooth vetch. Overall, our study provides a vital characterization of two full-length transcriptomes in Vicia villosa, which will be valuable for their molecular research and breeding. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with the acquisition of nosocomial infections, community-acquired infections, and infections related to livestock animals. In the pursuit of molecular targets in the development process of antibacterial drugs, enzymes within the shikimate pathway, such as 3-dehydroquinate dehydratase (DHQD), are regarded as promising targets. Therefore, through biochemical and biophysical techniques, in the present work, the characterization of DHQD from MRSA (SaDHQD) was performed. The kinetic results showed that the enzyme had a V_max of 107 μmol/min/mg, a K_m of 54 μM, a k_cat of 48 s⁻¹, and a catalytic efficiency of 0.9 μM⁻¹ s⁻¹. Within the biochemical parameters, the enzyme presented an optimal temperature of 55 °C and was thermostable at temperatures from 10 to 20 °C, being completely inactivated at 60 °C in 10 min. Furthermore, SaDHQD showed an optimal pH of 8.0 and was inactivated at pH 4.0 and 12.0. Moreover, the activity of the enzyme was affected by the presence of ions, surfactants, and chelating agents. The thermodynamic data showed that the rate of inactivation of the enzyme was a temperature-dependent process. Furthermore, the enthalpy change, entropy change, and Gibbs free energy change of inactivation were positive and practically constant, which suggested that the inactivation of SaDHQD by temperature was driven principally by enthalpic contributions. These results provide, for the first time, valuable information that contributes to the knowledge of this enzyme and will be useful in the search of SaDHQD inhibitors that can serve as leads to design a new drug against MRSA to combat antibiotic resistance. Full article

Bacteria resistance to antibiotics is a concerning global health problem; in this context, methicillin-resistant Staphylococcus aureus (MRSA) is considered as a high priority by the World Health Organization. Furthermore, patients with a positive result for COVID-19 received early antibiotic treatment, a fact that potentially encourages the increase in antibiotic resistance. Therefore, there is an urgency to develop new drugs with molecular mechanisms different from those of the actual treatments. In this context, enzymes from the shikimate pathway, a route absent in humans, such as dehydroquinate dehydratase (DHQD), are considered good targets. In this work, a computer-aided drug design strategy, which involved exhaustive virtual screening and molecular dynamics simulations with MM-PBSA analysis, as well as an in silico ADMETox characterization, was performed to find potential noncovalent inhibitors of DHQD from MRSA (SaDHQD). After filtering the 997 million compounds from the ZINC database, 6700 compounds were submitted to an exhaustive virtual screening protocol. From these data, four molecules were selected and characterized (ZINC000005753647 (1), ZINC000001720488 (2), ZINC000082049768 (3), and ZINC000644149506 (4)). The results indicate that the four potential inhibitors interacted with residues important for substrate binding and catalysis, with an estimated binding free energy like that of the enzyme’s substrate. Their ADMETox-predicted properties suggest that all of them support the structural characteristics to be considered good candidates. Therefore, the four compounds reported here are excellent option to be considered for future in vitro studies to design new SaDHQD noncovalent inhibitors and contribute to the search for new drugs against MRSA. Full article

Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu^®) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate E. coli producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor. Full article

Different southern grape (Muscadine) genotypes (Muscadinia rotundifolia Michx.) were evaluated for their contents of metabolites in ripe berries. The metabolome study identified 331 metabolites in ripening skin and seed tissues. The major chemical groups were organic acids, fatty acyls, polyketides, and organic heterocycle compounds. The metabolic pathways of the identified metabolite were mainly arginine biosynthesis, D-glutamine, D-glutamate metabolism, alanine, aspartate metabolism, aminoacyl-tRNA biosynthesis, and citrate cycle. Principal component analysis indicated that catechin, gallic acid, and epicatechin-3-gallate were the main metabolites existing in muscadine seed extracts. However, citramalic and malic acids were the main metabolites contributing to muscadine skin extracts. Partial least-squares discriminant analysis (VIP > 1) described 25 key compounds indicating the metabolome in muscadine tissues (skin and seed). Correlation analysis among the 25 compounds and oxidation inhibition activities identified five biomarker compounds that were associated with antioxidant activity. Catechin, gallic acid, epicatechin-3-gallate, fertaric acid, and procyanidin B1 were highly associated with DPPH, FRAP, CUPRAC, and ABTS. The five biomarker compounds were significantly accumulated in the seed relative to the skin tissues. An evaluation of 15 antioxidant-related genes represented by the 3-dehydroquinate dehydratase (DHD), shikimate kinase (SK), chalcone synthase (CHS), anthocyanidin reductase (ANR), laccase (LAC), phenylalanine ammonia-lyase (PAL), dihydroflavonol 4-reductase (DFR), 3-dehydroquinate synthase (DHQS), chorismate mutase (CM), flavanone-3-hydroxylase (F3H), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), leucoanthocyanidin reductase (LAR), gallate 1-β-glucosyltransferase (UGT), and anthocyanidin 3-O-glucosyltransferase (UFGT) encode critical enzymes related to polyphenolics pathway throughout four developmental stages (fruit-set FS, véraison V, ripe-skin R, and ripe-seed; S) in the C5 genotype demonstrated the dramatic accumulation of all transcripts in seed tissue or a developmental stage-dependent manner. Our findings suggested that muscadine grape seeds contain essential metabolites that could attract the attention of those interested in the pharmaceutical sector and the plant breeders to develop new varieties with high nutraceutical value. Full article

To study genetic variations between genomes of plants that are naturally tolerant and sensitive to glyphosate, we used two Zea mays L. lines traditionally bred in Poland. To overcome the complexity of the maize genome, two sequencing technologies were employed: Illumina and Single Molecule Real-Time (SMRT) PacBio. Eleven thousand structural variants, 4 million SNPs and approximately 800 thousand indels differentiating the two genomes were identified. Detailed analyses allowed to identify 20 variations within the EPSPS gene, but all of them were predicted to have moderate or unknown effects on gene expression. Other genes of the shikimate pathway encoding bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase and chorismate synthase were altered by variants predicted to have a high impact on gene expression. Additionally, high-impact variants located within the genes involved in the active transport of glyphosate through the cell membrane encoding phosphate transporters as well as multidrug and toxic compound extrusion have been identified. Full article

Plant anthocyanin biosynthesis is well understood, but the regulatory mechanism in purple foliage tea remains unclear. Using isobaric tag for relative and absolute quantification (iTRAQ), 815 differential proteins were identified in the leaves of Zijuan tea, among which 20 were associated with the regulation of anthocyanin metabolism. We found that the abundances of anthocyanin synthesis-related enzymes such as chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase and anthocyanin synthetase, as well as anthocyanin accumulation-related UDP-glucosyl transferase and ATP-binding cassette (ABC) transporters in the purple leaves were all significantly higher than those in the green leaves. The abundances of the transcription factors bHLH and HY5, regulating anthocyanin biosynthesis at transcriptional level were also obviously higher in purple leaves than those in green leaves. In addition, bifunctional 3-dehydroquinate dehydratase and chorismate mutase in purple leaves were distinctly higher in abundance compared to green leaves, which provided sufficient phenylalanine substrate for anthocyanin synthesis. Furthermore, lignin synthesis was found to be reduced due to the lower abundances of cinnamoyl-CoA reductase 1, peroxidase 15 and laccase-6, which resulted in increase of intermediates flow into anthocyanin synthesis pathway. The physiological data were consistent with proteomic results. These four aspects of biosynthetic regulation contribute to anthocyanin accumulation in purple leaves of Zijuan tea. Full article

The Paeonia root, with or without bark, are considered vital traditional Chinese medicine materials; the examples are those of Bai Shao, Chi Shao, and Dan Pi. In this study, we examine 24 genes and their expressions involved in the biosynthesis of paeoniflorin and its derivatives, which are active compounds of the Paeonia root, in Paeonia lactiflora and P. suffruticosa, as well as other related plants, Punica granatum, Rhus radicans, and Coriaria nepalensis. Our phylogenetic analyses suggest that these genes have functional diversity, and analysis of the transcriptional level shows paeoniflorin and gallic acid biosynthesis-related genes exhibit different transcription profiles in flowers, carpels, bark-free roots, and bark of P. lactiflora. The correlation analysis of gene expression and active compound contents support the idea that hydroxymethylglutaryl-CoA synthase and phosphomevalonate kinase in the mevalonate pathway and 3-dehydroquinate dehydratase/shikimate dehydrogenase in shikimate biosynthesis are potentially closely related to the accumulation of paeoniflorin and benzoylpaeoniflorin. Coupling gene diversity with chemical analysis, we show that paeoniflorin and its derived aromatic amino acids are predominant in bark. Full article