QSAR of 1,4-benzoxazin-3-one antimicrobials and their drug design perspectives.

Synthetic derivatives of 1,4-benzoxazin-3-ones have been shown to possess promising antimicrobial activity, whereas their natural counterparts were found lacking in this respect. In this work, quantitative structure-activity relationships (QSAR) of natural and synthetic 1,4-benzoxazin-3-ones as antimicrobials were established. Data published in literature were curated into an extensive dataset of 111 compounds. Descriptor selection was performed by a genetic algorithm. QSAR models revealed differences in requirements for activity against fungi, gram-positive and gram-negative bacteria. Shape, VolSurf, and H-bonding property descriptors were frequently picked in all models. The models obtained for gram-positive and gram-negative bacteria showed good predictive power (Q2Ext 0.88 and 0.85, respectively). Based on the models generated, an additional set of 1,4-benzoxazin-3-ones, for which no antimicrobial activity had been determined in literature, were evaluated in silico. Additionally, newly designed lead compounds with a 1,4-benzoxazin-3-one scaffold were generated in silico by varying the positions and combinations of substituents. Two of these were predicted to be up to 5 times more active than any of the compounds in the current dataset. The 1,4-benzoxazin-3-one scaffold was concluded to possess potential for the design of new antimicrobial compounds with potent antibacterial activity, a multitarget mode of action, and possibly reduced susceptibility to gram negatives' efflux pumps.

Effects of the antimicrobial glabridin on membrane integrity and stress response activation in Listeria monocytogenes.

Glabridin is a prenylated isoflavan which can be extracted from liquorice roots and has shown antimicrobial activity against foodborne pathogens and spoilage microorganisms. However, its application may be hindered due to limited information about its mode of action. In this study, we aimed to investigate the mode of action of glabridin using a combined phenotypic and proteomic approach on Listeria monocytogenes. Fluorescence and transmission electron microscopy of cells exposed to glabridin showed membrane permeabilization upon treatment with lethal concentrations of glabridin. Comparative proteomics analysis of control cells and cells exposed to sub-lethal concentrations of glabridin showed upregulation of proteins related to the two-component systems LiaSR and VirRS, confirming cell envelope damage during glabridin treatment. Additional upregulation of SigmaB regulon members signified activation of the general stress response in L. monocytogenes during this treatment. In line with the observed upregulation of cell envelope and general stress response proteins, sub-lethal treatment of glabridin induced (cross)protection against lethal heat and low pH stress and against antimicrobials such as nisin and glabridin itself. Overall, this study sheds light on the mode of action of glabridin and activation of the main stress responses to this antimicrobial isoflavan and highlights possible implications of its use as a naturally derived antimicrobial compound.

Switching the polarity of mouse enteroids affects the epithelial interplay with prenylated phenolics from licorice (Glycyrrhiza) roots.

The utility of 3D-small intestinal organoid (enteroid) models for evaluating effects of e.g. food (related) compounds is limited due to the apical epithelium facing the interior. To overcome this limitation, we developed a novel 3D-apical-out enteroid model for mice, which allows apical exposure. Using this model, we evaluated the effects on the enteroids' intestinal epithelium (including cytotoxicity, cell viability, and biotransformation) after exposure to glabridin, a prenylated secondary metabolite with antimicrobial properties from licorice roots (Glycyrrhiza glabra). Apical-out enteroids were five times less sensitive to glabridin exposure compared to conventional apical-in enteroids, with obtained cytotoxicities of 1.5 mM and 0.31 mM, respectively. Apical-out enteroids showed a luminal/apical layer of fucose rich mucus, which may contribute to the protection against potential cytotoxicity of glabridin. Furthermore, in apical-in enteroids IC50 values for cytotoxicity were determined for licochalcone A, glycycoumarin, and glabridin, the species-specific prenylated phenolics from the commonly used G. inflata, G. uralensis, and G. glabra, respectively. Both enteroid models differed in their functional phase II biotransformation capacity, where glabridin was transformed to glucuronide- and sulfate-conjugates. Lastly, our results indicate that the prenylated phenolics do not show cytotoxicity in mouse enteroids at previously reported minimum inhibitory concentrations (MICs) against a diverse set of Gram positive bacteria. Altogether, we show that apical-out enteroids provide a better mimic of the gastrointestinal tract compared to conventional enteroids and are consequently a superior model to study effects of food (related) compounds. This work revealed that prenylated phenolics with promising antibacterial activity show no harmful effects in the GI-tract at their MICs and therefore may offer a new perspective to control unwanted microbial growth.

Impact of food-relevant conditions and food matrix on the efficacy of prenylated isoflavonoids glabridin and 6,8-diprenylgenistein as potential natural preservatives against Listeria monocytogenes.

Prenylated isoflavonoids can be extracted from plants of the Leguminosae/Fabaceae family and have shown remarkable antimicrobial activity against Gram-positive food-borne pathogens, such as Listeria monocytogenes. Promising candidates from this class of compounds are glabridin and 6,8-diprenylgenistein. This research aimed to investigate the potential of glabridin and 6,8-diprenylgenistein as food preservatives against L. monocytogenes. Their antimicrobial activity was tested in vitro at various conditions relevant for food application, such as different temperatures (from 10 °C to 37 °C), pH (5 and 7.2), and in the presence or absence of oxygen. The minimum inhibitory concentrations of glabridin and 6,8-diprenylgenistein in vitro were between 0.8 and 12.5 µg/mL in all tested conditions. Growth inhibitory activities were similar at 10 °C compared to higher temperatures, although bactericidal activities decreased when the temperature decreased. Notably, lower pH (pH 5) increased the growth inhibitory and bactericidal activity of the compounds, especially for 6,8-diprenylgenistein. Furthermore, similar antimicrobial efficacies were shown anaerobically compared to aerobically at the tested conditions. Glabridin showed a more stable inhibitory and bactericidal activity when the temperature decreased compared to 6,8-diprenylgenistein. Therefore, we further determined the antimicrobial efficacy of glabridin against L. monocytogenes growth on fresh-cut cantaloupe at 10 °C. In these conditions, concentrations of glabridin of 50, 100 and 250 µg/g significantly reduced the growth of L. monocytogenes compared to the control, resulting on average in >1 Log CFU/g difference after 4 days compared to the control. Our results further underscored the importance of considering the food matrix when assessing the activity of novel antimicrobials. Overall, this study highlights the potential of prenylated isoflavonoids as naturally derived food preservatives.

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids.

Prenylated isoflavonoids are phytochemicals with promising antifungal properties. Recently, it was shown that glabridin and wighteone disrupted the plasma membrane (PM) of the food spoilage yeast Zygosaccharomyces parabailii in distinct ways, which led us to investigate further their modes of action (MoA). Transcriptomic profiling with Z. parabailii showed that genes encoding transmembrane ATPase transporters, including Yor1, and genes homologous to the pleiotropic drug resistance (PDR) subfamily in Saccharomyces cerevisiae were upregulated in response to both compounds. Gene functions involved in fatty acid and lipid metabolism, proteostasis, and DNA replication processes were overrepresented among genes upregulated by glabridin and/or wighteone. Chemogenomic analysis using the genome-wide deletant collection for S. cerevisiae further suggested an important role for PM lipids and PM proteins. Deletants of gene functions involved in biosynthesis of very-long-chain fatty acids (constituents of PM sphingolipids) and ergosterol were hypersensitive to both compounds. Using lipid biosynthesis inhibitors, we corroborated roles for sphingolipids and ergosterol in prenylated isoflavonoid action. The PM ABC transporter Yor1 and Lem3-dependent flippases conferred sensitivity and resistance, respectively, to the compounds, suggesting an important role for PM phospholipid asymmetry in their MoAs. Impaired tryptophan availability, likely linked to perturbation of the PM tryptophan permease Tat2, was evident in response to glabridin. Finally, substantial evidence highlighted a role of the endoplasmic reticulum (ER) in cellular responses to wighteone, including gene functions associated with ER membrane stress or with phospholipid biosynthesis, the primary lipid of the ER membrane. IMPORTANCE Preservatives, such as sorbic acid and benzoic acid, inhibit the growth of undesirable yeast and molds in foods. Unfortunately, preservative tolerance and resistance in food spoilage yeast, such as Zygosaccharomyces parabailii, is a growing challenge in the food industry, which can compromise food safety and increase food waste. Prenylated isoflavonoids are the main defense phytochemicals in the Fabaceae family. Glabridin and wighteone belong to this group of compounds and have shown potent antifungal activity against food spoilage yeasts. The present study demonstrated the mode of action of these compounds against food spoilage yeasts by using advanced molecular tools. Overall, the cellular actions of these two prenylated isoflavonoids share similarities (at the level of the plasma membrane) but also differences. Tryptophan import was specifically affected by glabridin, whereas endoplasmic reticulum membrane stress was specifically induced by wighteone. Understanding the mode of action of these novel antifungal agents is essential for their application in food preservation.

Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus.

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

Valorisation of liquorice (Glycyrrhiza) roots: antimicrobial activity and cytotoxicity of prenylated (iso)flavonoids and chalcones from liquorice spent (G. glabra, G. inflata, and G. uralensis).

Prenylated phenolics are antimicrobials found in liquorice (Glycyrrhiza spp.). Liquorice spent is a by-product rich in prenylated phenolics obtained after water extraction of roots, and is currently not valorised. We analysed the prenylated phenolics composition of spent extracts from Glycyrrhiza glabra, G. inflata, and G. uralensis, their antimicrobial activity, cytotoxicity, and effects on Caco-2 cell viability. G. glabra, G. inflata, and G. uralensis spent extracts showed distinct phytochemical profiles. Antibacterial activity (Lactobacillus buchneri, Streptococcus mutans, and Staphylococcus aureus) of G. uralensis and G. inflata (MICs 25-250 µg mL-1) was higher than of G. glabra (MICs 75-1000 µg mL-1). Marker compounds glabridin, licochalcone A, and glycycoumarin were equally potent (MICs 12.5-25 µg mL-1). G. inflata and G. uralensis showed cytotoxicity at 500 µg mL-1, whereas G. glabra was not toxic up to 1000 µg mL-1, but showed reduced viability between 50-500 µg mL-1. Linking antibacterial activity of the liquorice spent extracts with cell viability showed that MICs against S. aureus coincide with concentrations where cell viability was not reduced, whereas for the other bacteria and yeasts MICs concurred at concentrations where cell viability was reduced. In this study we show that liquorice spent is a by-product rich in antibacterial prenylated phenolics that offers interesting oppurtunities for e.g. control of microorganisms and the discovery of novel plant-derived antimicrobials.

Insights into the molecular properties underlying antibacterial activity of prenylated (iso)flavonoids against MRSA.

High resistance towards traditional antibiotics has urged the development of new, natural therapeutics against methicillin-resistant Staphylococcus aureus (MRSA). Prenylated (iso)flavonoids, present mainly in the Fabaceae, can serve as promising candidates. Herein, the anti-MRSA properties of 23 prenylated (iso)flavonoids were assessed in-vitro. The di-prenylated (iso)flavonoids, glabrol (flavanone) and 6,8-diprenyl genistein (isoflavone), together with the mono-prenylated, 4'-O-methyl glabridin (isoflavan), were the most active anti-MRSA compounds (Minimum Inhibitory Concentrations (MIC) ≤ 10 µg/mL, 30 µM). The in-house activity data was complemented with literature data to yield an extended, curated dataset of 67 molecules for the development of robust in-silico prediction models. A QSAR model having a good fit (R2adj 0.61), low average prediction errors and a good predictive power (Q2) for the training (4% and Q2LOO 0.57, respectively) and the test set (5% and Q2test 0.75, respectively) was obtained. Furthermore, the model predicted well the activity of an external validation set (on average 5% prediction errors), as well as the level of activity (low, moderate, high) of prenylated (iso)flavonoids against other Gram-positive bacteria. For the first time, the importance of formal charge, besides hydrophobic volume and hydrogen-bonding, in the anti-MRSA activity was highlighted, thereby suggesting potentially different modes of action of the different prenylated (iso)flavonoids.

A targeted prenylation analysis by a combination of IT-MS and HR-MS: Identification of prenyl number, configuration, and position in different subclasses of (iso)flavonoids.

Prenylated (iso)flavonoids are potent bioactive compounds found in the Fabaceae family. Analysis and quantification of this type of phytochemicals is challenging due to their large structural diversity. In this study, the fragmentation of prenylated (iso)flavonoids was investigated using electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) with fragmentation by collision induced dissociation (CID) in combination and Orbitrap-MS (ESI-FT-MS2) with fragmentation by higher energy C-trap dissociation (HCD). With this combination of IT-MSn and high resolution MS (FT-MSn), it was possible to determine the fragmentation pathways and characteristic spectral features of different subclasses of prenylated (iso)flavonoid standards, as well as characteristic fragmentations and neutral losses of different prenyl configurations. Based on our findings, a decision guideline was developed to (i) identify (iso)flavonoid backbones, (ii) annotate prenyl number, (iii) configuration, and (iv) position of unknown prenylated (iso)flavonoids, in complex plant extracts. In this guideline, structural characteristics were identified based on: (i) UV absorbance of the compound, (ii) mass-to-charge (m/z) ratio of the parent compound; (iii) ratio of relative abundances between neutral losses 42 and 56 u in MSn; (iv) retro-Diels-Alder (RDA) fragments, neutral losses 54 and 68 u, and the ratio [M+H-C4H8]+/[M+H]+. Using this guideline, 196 prenylated (iso)flavonoids were annotated in a Glycyrrhiza glabra root extract. In total, 75 skeletons were single prenylated, 104 were double prenylated, and for merely 17 skeletons prenyl number could not unambiguously be annotated. Our prenylation guideline allows rapid screening for identification of prenylated (iso)flavonoids, including prenyl number, configuration, and position, in complex plant extracts. This guideline supports research on these bioactive compounds in the areas of plant metabolomics and natural products.

Simultaneous Analysis of Glucosinolates and Isothiocyanates by Reversed-Phase Ultra-High-Performance Liquid Chromatography-Electron Spray Ionization-Tandem Mass Spectrometry.

A new method to simultaneously analyze various glucosinolates (GSLs) and isothiocyanates (ITCs) by reversed-phase ultra-high-performance liquid chromatography-electron spray ionization-tandem mass spectrometry has been developed and validated for 14 GSLs and 15 ITCs. It involved derivatization of ITCs with N-acetyl-l-cysteine (NAC). The limits of detection were 0.4-1.6 µM for GSLs and 0.9-2.6 µM for NAC-ITCs. The analysis of Sinapis alba, Brassica napus, and Brassica juncea extracts spiked with 14 GSLs and 15 ITCs indicated that the method generally had good intraday (≤10% RSD) and interday precisions (≤16% RSD). Recovery of the method was unaffected by the extracts and within 71-110% for GSLs and 66-122% for NAC-ITCs. The method was able to monitor the enzymatic hydrolysis of standard GSLs to ITCs in mixtures. Furthermore, GSLs and ITCs were simultaneously determined in Brassicaceae plant extracts before and after myrosinase treatment. This method can be applied to further investigate the enzymatic conversion of GSLs to ITCs in complex mixtures.

Induction of promising antibacterial prenylated isoflavonoids from different subclasses by sequential elicitation of soybean.

Elicited soybean (Glycine max (L.) Merrill, Leguminosae) seedlings can produce prenylated isoflavonoids from different subclasses, namely pterocarpans (glyceollins), isoflavones and coumestans. These prenylated isoflavonoids serve as defence compounds and can possess antimicrobial activity. Recently, we showed that priming with reactive oxygen species (ROS) specifically stimulated the production of glyceollins in Rhizopus spp.-elicited soybean seedlings (ROS + R). In this study, we achieved diversification of the inducible subclasses of prenylated isoflavonoids in soybean, by additional stimulation of two prenylated isoflavones and one prenylated coumestan. This was achieved by using a combination of the relatively long-lived ROS representative, H2O2, with AgNO3 prior to microbial elicitation. Microbial elicitation was performed with a live preparation of either a phytopathogenic fungus, Rhizopus spp. or a symbiotic bacterium, Bacillus subtilis. B. subtilis induced 30% more prenylated isoflavones than Rhizopus spp. in (H2O2 + AgNO3)-treated seedlings, without significantly compromising the total levels of glyceollins, compared to (ROS + R)-treated seedlings. The most abundant prenylated isoflavone induced was 6-prenyl daidzein, which constituted 60% of the total isoflavones. The prenylated coumestan, phaseol, was also induced in the (H2O2 + AgNO3)-treated and microbially elicited seedlings. Based on previously developed quantitative structure-activity relationship (QSAR) models, 6-prenyl daidzein and phaseol were predicted to be promising antibacterials. Overall, we show that treatment with H2O2 and AgNO3 prior to microbial elicitation leads to the production of promising antibacterial isoflavonoids from different subclasses. Extracts rich in prenylated isoflavonoids may potentially be applied as natural antimicrobial agents.

Enhanced biosynthesis of the natural antimicrobial glyceollins in soybean seedlings by priming and elicitation.

Glyceollins are a class of antimicrobial prenylated pterocarpans produced in soybean seedlings upon fungus elicitation. Priming with reactive oxygen species (ROS) prior to elicitation with Rhizopus oligosporus/oryzae (R) was investigated for its potential to enhance glyceollin production. ROS-priming prior to R-elicitation (ROS + R) increased glyceollin production (8.6 ± 0.9 µmol/g dry weight (DW)) more than 4-fold compared to elicitation without priming (1.9 ± 0.4 µmol/g DW). Furthermore, ROS-priming was superior to two physical primers which were used as benchmark primers, namely slicing (5.0 ± 0.6 µmol glyceollins/g DW) and sonication (4.8 ± 1.0 µmol glyceollins/g DW). Subsequently, the robustness of ROS + R was assessed by applying it to another soybean cultivar, where it also resulted in a significantly higher glyceollin content than R-elicitation without priming. ROS-priming prior to elicitation provides opportunities for improving the yield in large-scale production of natural antimicrobials due to the ease of application and the robustness of the effect across cultivars.

Modulation of Glucosinolate Composition in Brassicaceae Seeds by Germination and Fungal Elicitation.

Glucosinolates (GSLs) are of interest for potential antimicrobial activity of their degradation products and exclusive presence in Brassicaceae. Compositional changes of aliphatic, benzenic, and indolic GSLs of Sinapis alba, Brassica napus, and B. juncea seeds by germination and fungal elicitation were studied. Rhizopus oryzae (nonpathogenic), Fusarium graminearum (nonpathogenic), and F. oxysporum (pathogenic) were employed. Thirty-one GSLs were detected by reversed-phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MSn). Aromatic-acylated derivatives of 3-butenyl GSL, p-hydroxybenzyl GSL, and indol-3-ylmethyl GSL were for the first time tentatively annotated and confirmed to be not artifacts. For S. alba, germination, Rhizopus elicitation, and F. graminearum elicitation increased total GSL content, mainly consisting of p-hydroxybenzyl GSL, by 2-3 fold. For B. napus and B. juncea, total GSL content was unaffected by germination or elicitation. In all treatments, aliphatic GSL content was decreased (≥50%) in B. napus and remained unchanged in B. juncea. Indolic GSLs were induced in all species by germination and nonpathogenic elicitation.

Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids.

Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L-1 (0.35 µM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.

Rapid membrane permeabilization of Listeria monocytogenes and Escherichia coli induced by antibacterial prenylated phenolic compounds from legumes.

Prenylated phenolics from the Fabaceae are promising lead compounds for new antibacterials. Pools enriched in prenylated phenolics were made from lupine, peanut and soybean seedlings. One pool was rich in chain prenylated isoflavones (cIsf), one in chain prenylated stilbenoids (cSti), one in chain prenylated (cPta) and one in ring-closed prenylated pterocarpans (rPta), as characterized by RP-UHPLC-UV-MS. Antibacterial activity of the pools and membrane permeabilization was investigated. Pools showed high antibacterial activity against Listeria monocytogenes: cIsf pool had a minimum inhibitory concentration of 10µg/ml prenylated compounds, followed by cPta pool (25µg/ml) and cSti pool (35µg/ml). Activity against E. coli was found only when the pools were co-administered with an efflux pump inhibitor. The pool enriched in chain prenylated isoflavones permeabilized the bacterial membrane within minutes of exposure, whereas ampicillin did not. Bent conformation and chain prenylation, were molecular features of main prenylated phenolics found in pools with high antibacterial activity.

QSAR-based molecular signatures of prenylated (iso)flavonoids underlying antimicrobial potency against and membrane-disruption in Gram positive and Gram negative bacteria.

Prenylated flavonoids and isoflavonoids are phytochemicals with remarkable antibacterial activity. In this study, 30 prenylated (iso)flavonoids were tested against Listeria monocytogenes and Escherichia coli (the latter in combination with an efflux pump inhibitor). Minimum inhibitory concentrations of the most active compounds ranged between 6.3-15.0 µg/mL. Quantitative structure-activity relationships (QSAR) analysis was performed and linear regression models were proposed with R2 between 0.77-0.80, average R2m between 0.70-0.75, Q2LOO between 0.66-0.69, and relatively low amount of descriptors. Shape descriptors (related to flexibility and globularity), together with hydrophilic/hydrophobic volume and surface area descriptors, were identified as important molecular characteristics related to activity. A 3D pharmacophore model explaining the effect of the prenyl position on the activity of compounds was developed for each bacterium. These models predicted active compounds with an accuracy of 71-88%. With regard to the mode of action, good antibacterial prenylated (iso)flavonoids with low relative hydrophobic surface area caused remarkable membrane permeabilization, whereas those with higher relative hydrophobic surface area did not. Based on the QSAR and membrane permeabilization studies, the mode of action of antibacterial prenylated (iso)flavonoids was putatively rationalized.

The position of prenylation of isoflavonoids and stilbenoids from legumes (Fabaceae) modulates the antimicrobial activity against Gram positive pathogens.

The legume plant family (Fabaceae) is a potential source of antimicrobial phytochemicals. Molecular diversity in phytochemicals of legume extracts was enhanced by germination and fungal elicitation of seven legume species, as established by RP-UHPLC-UV-MS. The relationship between phytochemical composition, including different types of skeletons and substitutions, and antibacterial properties of extracts was investigated. Extracts rich in prenylated isoflavonoids and stilbenoids showed potent antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus at concentrations between 0.05 and 0.1% (w/v). Prenylated phenolic compounds were significantly (p<0.01) correlated with the antibacterial properties of the extracts. Furthermore, the position of the prenyl group within the phenolic skeleton also influenced the antibacterial activity. Overall, prenylated phenolics from legume seedlings can serve multiple purposes, e.g. as phytoestrogens they can provide health benefits and as natural antimicrobials they offer preservation of foods.

Structural basis for non-genuine phenolic acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase CloQ from the ABBA/PT-barrel superfamily.

Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.