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Methods in Enzymology 2024Terpene synthases (TS) transform achiral prenyl substrates into elaborate hydrocarbon scaffolds with multiple stereocenters through a series of cyclization reactions and...
Terpene synthases (TS) transform achiral prenyl substrates into elaborate hydrocarbon scaffolds with multiple stereocenters through a series of cyclization reactions and carbon skeleton rearrangements. The reactions involve high-energy carbocation intermediates that must be stabilized by the enzyme along the pathway to the desired products. A variety of substrate analogs have been used to investigate TS mechanism. This article will focus on a class of analogs which strategically replace hydrogen atoms with fluorine to inhibit the generation of specific carbocation intermediates. We will explore the synthesis and use of the analogs to study TS mechanism.
Topics: Alkyl and Aryl Transferases; Substrate Specificity; Cyclization; Terpenes
PubMed: 38942503
DOI: 10.1016/bs.mie.2024.04.003 -
Methods in Enzymology 2024The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have...
The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have been devoted towards increasing metabolic flux catalyzed by terpene synthases, employing strategies such as gene overexpression and protein engineering. Notably, numerous studies have demonstrated remarkable titer improvements by applying translational fusion, typically by fusing the terpene synthase with a prenyl diphosphate synthase that catalyzes the preceding step in the pathway. The main appeal of the translational fusion approach lies in its simplicity and orthogonality to other metabolic engineering tools. However, there is currently limited understanding of the underlying mechanism of flux enhancement, owing to the unpredictable and often protein-specific effects of translational fusion. In this chapter, we discuss practical considerations when engineering translationally fused terpene synthases, drawing insights from our experience and existing literature. We also provide detailed experimental workflows and protocols based on our previous work in budding yeast (Saccharomyces cerevisiae). Our intention is to encourage further research into the translational fusion of terpene synthases, anticipating that this will contribute mechanistic insights not only into the activity, behavior, and regulation of terpene synthases, but also of other enzymes.
Topics: Alkyl and Aryl Transferases; Metabolic Engineering; Saccharomyces cerevisiae; Recombinant Fusion Proteins; Terpenes; Protein Biosynthesis; Protein Engineering
PubMed: 38942501
DOI: 10.1016/bs.mie.2024.02.005 -
Molecules (Basel, Switzerland) Jun 2024Propolis is a resinous bee product with a very complex composition, which is dependent upon the plant sources that bees visit. Due to the promising antimicrobial...
Propolis is a resinous bee product with a very complex composition, which is dependent upon the plant sources that bees visit. Due to the promising antimicrobial activities of red Brazilian propolis, it is paramount to identify the compounds responsible for it, which, in most of the cases, are not commercially available. The aim of this study was to develop a quick and clean preparative-scale methodology for preparing fractions of red propolis directly from a complex crude ethanol extract by combining the extractive capacity of counter-current chromatography (CCC) with preparative HPLC. The CCC method development included step gradient elution for the removal of waxes (which can bind to and block HPLC columns), sample injection in a single solvent to improve stationary phase stability, and a change in the mobile phase flow pattern, resulting in the loading of 2.5 g of the Brazilian red propolis crude extract on a 912.5 mL Midi CCC column. Three compounds were subsequently isolated from the concentrated fractions by preparative HPLC and identified by NMR and high-resolution MS: red pigment, retusapurpurin A; the isoflavan 3()-7-O-methylvestitol; and the prenylated benzophenone isomers xanthochymol/isoxanthochymol. These compounds are markers of red propolis that contribute to its therapeutic properties, and the amount isolated allows for further biological activities testing and for their use as chromatographic standards.
Topics: Propolis; Countercurrent Distribution; Chromatography, High Pressure Liquid; Brazil; Animals; Chemical Fractionation; Bees
PubMed: 38930823
DOI: 10.3390/molecules29122757 -
Foods (Basel, Switzerland) Jun 2024The Food and Agricultural Organization estimates a 17% loss in the food production chain, making it imperative to adopt scientific and technological approaches to...
The Food and Agricultural Organization estimates a 17% loss in the food production chain, making it imperative to adopt scientific and technological approaches to address this issue for sustainability. Industrial food production waste and its value-added applications, particularly in relation to a wide variety of pathogenic microorganisms and the health-related effects have not been thoroughly investigated. This study explores the potential of food production waste extracts-lemon peel (LP), hot trub (HT), and coffee silverskin (CSS) as sources of bioactive compounds. Extraction was conducted using hydro-methanolic extraction with yields in LP (482 mg/1 g) > HT (332 mg/1 g) > CSS (20 mg/1 g). The agar diffusion assay revealed the substantial antibacterial activity of all three extracts against , , , and . All extracts demonstrated activity against Gram-positive and Gram-negative bacteria, displaying minimum inhibitory concentrations effective against pathogenic bacteria like , , , and . Total phenolic content (TPC in mg GAE/1g) was 100, 20, and 100 for CSS, HT, and LP, respectively. Antioxidant activity by ABTS indicated IC of 3.09, 13.09, and 2.61 for LP, HT, and CSS, respectively. Also, the antioxidant activity of the extracts was further confirmed by DPPH assay with the best activity in CSS (9.84 GAEg) and LP (9.77 mg of GAEg) rather than in HT (1.45 GAEg). No adverse cytotoxic effects on HaCaT cells were observed. Pancreatic amylase inhibition demonstrated antidiabetic potential, with LP showing the highest levels (92%). LC-MS characterization identified polyphenols as the main compounds in CSS, prenylated compounds in HT, and flavanols in LP. The findings imply the potential sustainable use of food production waste in industry.
PubMed: 38928843
DOI: 10.3390/foods13121902 -
Chemistry & Biodiversity Jun 2024Three naturally occurring prenylated pyranocoumarins, nordentatin (1), dentatin (2), and clausarin (3), isolated from the roots of Clausena excavata (Family Rutaceae),...
Three naturally occurring prenylated pyranocoumarins, nordentatin (1), dentatin (2), and clausarin (3), isolated from the roots of Clausena excavata (Family Rutaceae), and O-methylclausarin (4) which was obtained by methylation of 3, were investigated for their α-glucosidase inhibitory activity. The mechanism of action and the in silico prediction of their physicochemical and ADMET properties as well as the molecular docking were also studied. Compounds 1-4 exhibited stronger α-glucosidase inhibitory activity than the positive control, acarbose, through a non-competitive mechanism. Among them, 3 exhibited the highest activity, with an IC50 of 8.36 µM, which is significantly stronger than that of acarbose (IC50 = 430.35 µM). The prenyl group on C-3 and the hydroxyl group on C-5 in 3 may play important roles in enhancing the activity. Calculated physicochemical and ADMET parameters of 1-4 satisfied the Lipinski's and Veber's rules. Molecular simulation analysis indicated they are promising drug candidates with no hepatotoxicity. Compound 3 exhibited potent activity in the experiment and demonstrated good drug properties based on the calculations. A molecular docking study revealed that 3 showed H-bonding and π-π stacking interactions with selective Phe321, as well as interactions with thirteen other amino acid residues of the α-glucosidase.
PubMed: 38923383
DOI: 10.1002/cbdv.202401141 -
Cells Jun 2024Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs)....
Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of , the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in cells by transduction with gene replacement (ShH10-CMV-) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.
Topics: Humans; Adaptor Proteins, Signal Transducing; Autophagy; Choroideremia; Induced Pluripotent Stem Cells; Mechanistic Target of Rapamycin Complex 1; Models, Biological; rab GTP-Binding Proteins; Retinal Pigment Epithelium; Signal Transduction
PubMed: 38920696
DOI: 10.3390/cells13121068 -
Natural Product Research Jun 2024A detailed study on secondary metabolites from the stem bark of has yielded one triterpenoid and four xanthones. Along with that, five novel rubraxanthone derivatives...
A detailed study on secondary metabolites from the stem bark of has yielded one triterpenoid and four xanthones. Along with that, five novel rubraxanthone derivatives had been successfully synthesised Williamson etherification with various alkyl halides. The antibacterial evaluation on crude extract, isolated secondary metabolites , and synthesised compounds against , and demonstrated moderate to active activities outlining their bacteriostatic potential. The structure-activity relationship (SAR) study conducted revealed the presence of prenyl and hydroxy groups on the xanthone attributed to good bacterial inhibition. The introduction of the alkyl chain to the hydroxy part eventually decreases the antibacterial activity of the compound which is probably due to the bulkiness that causes steric hindrances, therefore limiting the ability to bind to its target site within the bacterial cell.
PubMed: 38919065
DOI: 10.1080/14786419.2024.2371109 -
RSC Advances Jun 2024The enzymatic decarboxylation of α,β-unsaturated acids using the ferulic acid decarboxylase (Fdc1) enzyme and prenylated flavin mononucleotide (prFMN) cofactor is a...
The enzymatic decarboxylation of α,β-unsaturated acids using the ferulic acid decarboxylase (Fdc1) enzyme and prenylated flavin mononucleotide (prFMN) cofactor is a potential, environmentally friendly reaction for the biosynthesis of styrene and its derivatives. However, experiments showed that the enzyme activity of Fdc1 depends on the ring structure of prFMN, namely, the iminium and ketimine forms, and the loss of enzyme activity results from prFMN → prFMN photoisomerization. To obtain insight into this photochemical process and to improve the enzyme efficiency of Fdc1, two proposed photoisomerization mechanisms with different proton sources for the acid-base reaction were studied herein using theoretical methods. The potential energy surfaces calculated using the density functional theory method with the Becke, 3-parameter, and Lee-Yang-Parr hybrid functionals and DZP basis set (DFT/B3LYP/DZP) and TD-DFT/B3LYP/DZP methods confirmed that the light-dependent reaction occurs in the rate-determining proton transfer process and that the mechanism involving intermolecular proton transfer between prFMN and Glu282 (external base) is energetically more favorable than that involving intramolecular proton transfer in prFMN (internal base). The thermodynamic results obtained from the transition state theory method suggested that the exothermic relaxation energy in the photo-to-thermal process can promote the spontaneous formation of a high-energy-barrier transition state, and an effective enzymatic decarboxylation could be achieved by slowing down the formation of the undesirable thermodynamically favorable product (prFMN). Because the rate constant for formation of the high-energy-barrier transition state varies exponentially over the temperature range of 273-298 K, and experimental results have shown that incubating Fdc1 on ice results in a complete loss of enzyme activity, it is recommended to perform the decarboxylation reaction at 285 K to strike a balance between minimizing enzyme stability loss at 273 K and mitigating the effects of UV irradiation. The computational strategy and fundamental insights obtained in this study could serve as guidelines for future theoretical and experimental investigations on the same and similar photochemical systems.
PubMed: 38915324
DOI: 10.1039/d4ra02035a -
Journal of Ethnopharmacology Jun 2024Plants have been used for a long time in traditional medicine to treat many diseases. The genus Prangos belongs to the Apiaceae family and has various medicinal and... (Review)
Review
ETHNOPHARMACOLOGICAL RELEVANCE
Plants have been used for a long time in traditional medicine to treat many diseases. The genus Prangos belongs to the Apiaceae family and has various medicinal and aromatic species. Since ancient times, Prangos species have been employed extensively in traditional medicine for different purposes and are especially popular for their aphrodisiac effects.
AIM OF THE REVIEW
The goal of this paper is to represent a systematic review of the species in the genus Prangos, including their botanical characteristics, uses in traditional medicine, phytochemical constituents, the composition of the essential oils produced, and the biological properties.
MATERIALS AND METHODS
The articles and keywords regarding traditional uses and bioactivities of Prangos species were evaluated using electronic databases such as PubMed, Google Scholar, and ScienceDirect. Use of the World Flora Online (WFO) - The Plant List, The International Plant Names Index, the World Checklist of Vascular Plants (2024), and ChemDraw Professional helped complete this compilation.
RESULTS
Phytochemical investigations have indicated that coumarins are characteristic constituents of Prangos species, especially prenylated and furanocoumarins, and also flavonoids, terpenoids, and phytosterols occur in this genus. In addition, the essential oils of these plants have been examined. The biological properties of the Prangos species seem worthy of further investigation. Also, some information about the toxicity of these species and their use as ingredients in food products is presented.
CONCLUSIONS
This review highlights the evaluation of traditional knowledge, phytochemical profiles, biological activities, and potential uses of Prangos species as foods and spices. Many pharmacological activities have been performed related to their traditional uses, but frequently, the exact mechanism of action remains scientifically unproven. This review has compiled data on the phytochemistry, the active secondary metabolites, the biological properties, and recent advances in Prangos species.
PubMed: 38909827
DOI: 10.1016/j.jep.2024.118480 -
Journal of Agricultural and Food... Jun 2024The overuse of antibiotics in animal farming and aquaculture has led to multidrug-resistant methicillin-sensitive (MR-MSSA) becoming a common pathogen in foodborne...
The overuse of antibiotics in animal farming and aquaculture has led to multidrug-resistant methicillin-sensitive (MR-MSSA) becoming a common pathogen in foodborne diseases. Ait. serves as a traditional plant antibacterial agent and functional food ingredient. A total of 30 compounds (-) were isolated from the root bark of , consisting of 20 new compounds (-). In the biological activity assay, compound demonstrated a remarkable inhibitory effect on MR-MSSA, with an MIC of 2 μg/mL. Furthermore, was found to rapidly eliminate bacteria, inhibit biofilm growth, and exhibit exceptionally low cytotoxicity. Mechanistic studies have revealed that possesses an enhanced membrane-targeting ability, binding to the bacterial cell membrane components phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL). This disruption of bacterial cell membrane integrity increases intracellular reactive oxygen species, protein and DNA leakage, reduced bacterial metabolism, and ultimately bacterial death. In summary, these findings suggest that compound holds promise as a lead compound against MR-MSSA.
PubMed: 38905352
DOI: 10.1021/acs.jafc.4c01430