Frontiers in Chemistry

Gastrointestinal infections are a leading cause of death and morbidity worldwide. Antibiotics as current treatments are ineffective or not recommended due to the emergence of multidrug resistant organisms. The initial step of the pathogenesis for most of the enteric infections is the binding of the pathogen to oligosaccharide receptors on the surface of host cells, making these receptors attractive targets for novel antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed an alternative therapeutic intervention based on a novel microbiome-based chemical engineering approach. We synthesized cholesteryl-peptides as flexible chemical scaffold for the presentation of carbohydrate GM1 mimic receptors in probiotic bacteria acting as a decoy for cholera toxin neutralization. The GM1-cholesteryl-peptides were stably embedded into the membranes of both gram positive and gram negative probiotics, Lactobacillus reuteri DSM and Escherichia coli Nissle 1917 respectively. The resulting GM1-coated probiotics demonstrated to quench nearly 100% of cholera toxin in vitro, functioning as decoys by preventing toxin binding to epithelial cells. The present study strongly suggests that our new and versatile biomimetic non-recombinant system could be employed to neutralize the functional effect of infections mediated by oligosaccharide receptors.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19F-labeling system is still lacking, including an effective 19F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)-ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19F-probe-profiled conformational states, as well as the rigidity and compactness of -CF3 in the target protein.

Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to selectively sulfate specific positions of various metabolites. To this end we need to understand the relation between sequence, structure and function. Actually, eukaryotic PAPS dependent sulfotransferases have extensively been studied, while we largely lack knowledge about the diversity of bacterial PAPS-independent ASSTs. We have conducted a multidisciplinary study of a bacterial sulfotransferase from Salmonella typhimurium, revealing the importance of active site residues for activity and guiding substrate specificity. Based on the crystal structure in complex with substrate, we used molecular docking combined with Quantum Mechanics/Molecular Mechanics (QM/MM) free energy calculations to investigate the reaction catalytic mechanism with a focus on the role of specific active site residues in substrate specificity. Our results show that the catalytic mechanism involving three conserved histidines is common with previously determined ASSTs, and highlight a specific pair of aromatic residues, Phe174 and Tyr557, that play an important role in defining the substrate specificity in ASST clade 5. These findings enhance our general understanding of how substrate specificity is attained and has the potential to guide further enzyme engineering to expand the acceptor scope of ASSTs.

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

In 1998 we predicted percent human intestinal absorption (HIA) for 86 drug and drug-like compounds from six molecular descriptors chosen by a genetic algorithm with a small neural network serving as the fitness function. That result rested on one division of the compounds into three sets: training, cross-validation and external prediction. Repeating the division shows it was a rather fortunate one. An extraction is one random draw from a wide distribution, so we repeat every experiment 250 times and report the result as a distribution. Across 250 stratified extractions, 14 match the published cross-validation error and only one matches the published external prediction error. Reconstructed this way, the published six descriptors give a mean extraction error roughly twice the external prediction value we reported in 1998 (Table 1). We then applied the same six descriptors to a larger set of 780 compounds from a public compilation. Under the 1998 network they give 22.50 %HIA units on training compounds against 25.77 on extracted compounds, whereas on the 86 they give 13.43 against 30.39. More compounds close the gap between fitting and prediction. Four datasets are modeled here: 780 small molecules with HIA measured in man, 3,771 small molecules and 1,281 cyclic peptides measured across Caco-2 cell monolayers, and 7,298 cyclic peptides measured across artificial membranes. Each is its own prediction problem, and one procedure is applied to all four. A genetic algorithm chooses which descriptors to use and how many, and a random forest grades each choice on compounds its own trees never saw. The descriptors chosen most often across the 250 extractions form one consensus set for each dataset. For absorption, 25 consensus descriptors under a random forest reach an extraction rms of 20.47 %HIA units at R 0.76. On the same compounds and the same extractions, the six 1998 descriptors under the 1998 network reach 25.77 at R 0.59. The four models are served at predicthia.ai, where a structure can be drawn or pasted, scored against its training set for similarity, and the training data with fitted models downloaded. Each model served is its fixed consensus set fitted on every compound in its dataset; the accuracy quoted beside a prediction comes from evaluating that same set across 250 extractions (Table 3). The fixed sets hold 25 descriptors for absorption, 55 for Caco-2 small-molecule permeability, 50 for Caco-2 peptide permeability and 25 for artificial-membrane permeability. Keywords: human intestinal absorption; QSPR; genetic algorithm; descriptor selection; cyclic peptides; membrane permeability; oral bioavailability; repeated extraction

This study aims to identify potential inhibitors of the Ag85C enzyme from Mycobacterium tuberculosis through primary docking, pharmacophore modelling, and ADME/pharmacokinetics analysis. Delamanid and Pretomanid emerged as the top hits in primary docking, demonstrating strong binding affinities and interactions with key active site residues. A robust pharmacophore model was developed, identifying essential features for binding and used to screen 4254 antituberculotic compounds, yielding three promising candidates: F0314-0040, F0398-0505, and F0349-2716. These compounds exhibited high binding affinities in major docking studies. ADME and pharmacokinetics analysis revealed favorable Drug likeness and absorption profiles, supporting their potential as effective antituberculotic agents. The integration of computational methods provided a comprehensive approach for rational drug design.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Proteolysis-targeting chimeras (PROTACs) are large, flexible molecules that frequently extend beyond conventional drug-like chemical space, including Lipinski's rule-of-five boundaries. Their membrane permeability has often been linked to molecular chameleonicity and the ability to adopt compact conformations with reduced polar surface exposure; however, the underlying conformational determinants remain unclear. Here, we investigated 20 PROTACs spanning distinct passive-permeability classes using enhanced-sampling molecular dynamics (ES-MD). All permeability classes accessed compact states with low radius of gyration (Rg) and polar surface area (PSA), indicating that compact-state accessibility alone does not distinguish permeable from non-permeable PROTACs. We therefore introduce maximum ring size (MRS), defined as the size of the largest intramolecular hydrogen-bonded ring, as a metric for identifying compact conformations stabilized by long-range intramolecular hydrogen bonding. Like the other descriptors examined, MRS neither separates nor ranks the permeability classes, but helps locate folded states in the large ensemble. Representative compact conformations from selected highly permeable, moderately permeable, and non-permeable compounds were subsequently examined by all-atom unbiased MD in water and chloroform. The highly permeable PROTAC held a closed-flat ensemble in both environments, but in two structurally distinct arrangements, whereas the other two redistributed across heterogeneous ensembles. Passive diffusion therefore appears to depend on the population, stability, and environment-dependent persistence of permeable-like states rather than on compactness alone.

Computational safety evaluation of phytochemical libraries commonly relies on models trained on experimentally tested compounds and applied to much larger and chemically diverse collections. An important but often overlooked question is whether predicted compounds are adequately represented within the experimental chemical space underlying these models. Here, we examined this question for the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database using experimentally characterized datasets for three anti-target endpoints: hERG blockade, cytochrome P450 (CYP) inhibition, and pregnane X receptor (PXR) induction. After structure standardization, 14,956 unique IMPPAT compounds were compared with 8,877 hERG, 4,905 CYP, and 4,138 PXR reference compounds using consensus nearest-neighbor similarity based on Morgan and atom-pair fingerprints. At a Tanimoto similarity threshold of 0.80, only 0.92%, 0.30%, and 0.46% of IMPPAT compounds had close measured analogs in the hERG, CYP, and PXR datasets, respectively. Relative to the internal nearest-neighbor distributions of the reference datasets, a typical IMPPAT compound lay near the 1st percentile, indicating substantial chemical-space separation. Compounds with close experimental analogs also showed distinct molecular characteristics, with descriptor-based logistic regression yielding cross-validated AUC values of 0.77-0.80. Plant-level enrichment was markedly reduced after excluding widely distributed metabolites and was further restricted when the nearest measured analog was required to have pIC50 or pEC50 [≥] 5. The remaining signals were concentrated in a limited number of taxa, including Rauvolfia, Sinopodophyllum/Dysosma, Cinchona, and Catharanthus roseus. Overall, most IMPPAT phytochemicals lack close structural representation among currently measured anti-target compounds. Computational safety predictions for these molecules should therefore be interpreted as extrapolations and accompanied by explicit measures of structural support. Absence of a close experimental analog should be regarded as a gap in available evidence rather than evidence of safety.

Exploring the ability of metabolic enzymes to interact with diverse small molecules can provide insights into their ligand recognition and activity regulation. Glucose-6-phosphate isomerase (GPI) is an enzyme that catalyzes the second step of glycolysis, a critical node that links glucose metabolism to other cytosolic pathways, such as pentose phosphate and hexosamine biosynthetic pathways. Despite its importance in cellular metabolism, little is known about the broad ligandability of GPI. Here, we performed a structure-based virtual screening using molecular docking to identify new modulators predicted to engage with the human GPI active site. Enzyme kinetic and binding studies confirmed eleven compounds as active-site binders of GPI, exhibiting inhibitory activities and binding affinities in upper micromolar to sub-millimolar range. The binding of several ligands to GPI active site was further supported by X-ray crystallographic data. In addition, in vitro assays were performed to evaluate the modulatory effects of the GPI hits on cellular glycolysis and viability. The identified inhibitors included the mono-phosphorylated metabolite of ribavirin, which inhibited glycolytic flux and cell viability; two compounds with phosphate bioisosteres; and endogenous phosphate-containing metabolites from the pentose phosphate, hexosamine, nucleotide, and mevalonate pathways. Collectively, these findings expanded the repertoire of human GPI ligands and revealed previously unrecognized GPI-metabolite interactions that may contribute to modulation of glycolytic flux.

Microplastic pollution from homopolymers such as polypropylene (PP), polystyrene (PS), and polyethylene (PE) poses growing health and environmental concerns, highlighting the need for robust polymer-degrading biocatalysts. We previously identified a naturally occurring cysteine-dependent hydrolase (CsdH) from Rhodococcus opacus, featuring a unique Cys160-Lys127-Asp39 catalytic triad and providing a novel mechanism for polyethylene terephthalate (PET) degradation. In this study, we have determined crystal structures of CsdH in complex with PP, PS, PE, and styrene and demonstrated the potential of the enzyme to directly degrade these homopolymers into their respective low molecular weight degradation products using LC-MS. Structural analysis reveals that the enzyme retains a conserved principal access tunnel while undergoing subtle substrate-dependent changes to accommodate different substrates. The enzyme exhibited broad plastic degradation activity with the highest weight loss observed for PE, followed by PP and PS. SEM analysis reveals surface roughness, cracks and grooves, confirming polymer degradation. Further, these results corroborate through FTIR and TGA analysis. These findings establish a strong foundation for enzyme engineering and aim at developing efficient microplastic degradation.

Albumin binding can substantially alter the unbound concentration and apparent cellular potency of small-molecule anticancer agents. BAY-1816032 is a selective BUB1 kinase inhibitor with activity in malignant pleural mesothelioma, but its interaction with serum albumin has not been defined. This study combined intrinsic bovine serum albumin (BSA) fluorescence measurements, molecular docking, and cell-viability assays to examine whether albumin association influences BAY-1816032 activity. BAY-1816032 produced pronounced quenching of BSA fluorescence at low drug-to-protein molar ratios, and the quenching response was greater at mildly acidic pH than at mildly basic pH. Docking to BSA (PDB 4F5S) placed the top-ranked pose in Sudlow site I (subdomain IIA), with a Vina score of -7.8 kcal/mol and predicted hydrogen-bond contacts with Tyr149, Arg256, and Asp450. In NCI-H28, NCI-H2052, NCI-H2452, and AB12 mesothelioma cells, supplementation of 10% fetal bovine serum medium with 2% (w/v; 20 mg/mL) BSA shifted BAY-1816032 concentration-response curves to the right. Relative to 10% serum alone, IC50 values increased from 2.4 to 11.1 uM in H28, 3.6 to 22.0 uM in H2052, 2.0 to 4.9 uM in H2452, and 2.4 to 11.1 uM in AB12. These data support an albumin-dependent reduction in the freely available drug fraction and identify site I as the leading computational binding-site hypothesis. Because fluorescence-intensity and docking data do not independently establish a dissociation constant or quenching mechanism, direct unbound-fraction measurements and competitive displacement experiments are required for quantitative confirmation.

Carbohydrates are essential molecules involved in a wide range of biological processes. While their structural representation in the Protein Data Bank (PDB) has been hindered due to their highly diverse and flexible nature, advancements on X-ray crystallography and CryoEM are accelerating the obtaining of their experimental structures, which in turn supports improved analyses of their structural properties, such as the role of their non-chair conformations on biological processes. In this context, the current work provides a comprehensive and up to date overview of carbohydrate structures within the PDB through classification, quantification, and characterization of monosaccharides and glycan chains, incorporating Cremer-Pople puckering coordinates to evaluate pyranose conformations. Our findings show that, even with a steep increase in CryoEM structures including more complex oligosaccharides, carbohydrate data remains concentrate dinsmallglycanchains, with a about 4% of non-chair conformation sintopresolution structures. Free energy landscapes for the pyranose ring in the presence and absence of nine different binding proteins, including both enzymes and non-enzymes, reinforce the preferred stability of chairs and the rarity of conformational induction upon binding.

Aromatic side chains carry the optical and redox chemistry of peptides, and lengthening one is widely expected to extend its conjugation and narrow its HOMO-LUMO gap. We test that expectation with plane-wave density functional theory on 22 isolated neutral peptides NH2-(X)n-COOH (X = Phe, Tyr, Trp, Cys; n = 1 to 5, and n = 10 for Cys and Trp). The gap does narrow, at the same rate for all three aromatic residues, i.e., -0.17 to -0.18 eV per residue, even though benzene, phenol and indole are very different chromophores. Residue-resolved projections reveal why: nothing delocalises. Every aromatic frontier orbital is 97 to 100 % confined to a single residue and the HOMO participation number saturates at one ring. Each residue instead keeps its own local gap: for Trp it is invariant to within 0.03 eV over n = 2 to 10, while over that same span the global gap falls by 1.19 eV. The global gap is therefore an extreme-value functional of the residue site energies, gap(n) =<g_f>-{Delta}(n), whose alignment penalty accounts for the entire effect: the polar backbone generates a monotonic internal electrostatic ramp that leaves the two gap edges at opposite ends of the chain, 32 [A] apart. We set out the resulting design rules for peptide-based bio-organic semiconductors.

Allosteric modulation of G protein-coupled receptors (GPCR) is a rapidly advancing area of drug discovery with considerable therapeutic potential. In this study, we used various bioluminescence resonance energy transfer (BRET) assay configurations to investigate the differential activity of LY3154207, a dopamine D1 receptor (D1R)- positive allosteric modulator (PAM), in potentiating D1R-mediated signaling pathways. Our data showed that LY3154207 demonstrated clear bias toward {beta}-arrestin recruitment over G-protein engagement. Among the G-protein subtypes, LY3154207 preferentially potentiated Gs signaling induced by DA. Interestingly, when tested for its effect on the Gs-selective D1R agonist DHX and the Golf-selective agonist TAV, LY3154207 further enhanced the selectivity of these compounds, suggesting a probe-dependent effect. We also studied the structural mechanism underlying this subtype selectivity and found that W123 and R130 in the intracellular loop 2 (ICL2) of D1R were crucial for the activity of LY3154207. Furthermore, the N domain of the G-protein appeared to make minor contributions to this G-protein subtype selectivity of LY3154207. Lastly, our data demonstrated that LY3154207 induced cAMP production in the absence of the orthosteric ligand, indicating that this compound is an ago-PAM.

The classical estrogen receptor (ER) is a steroid hormone receptor known for its role in both health and disease. ER, like its counterpart ER{beta}, acts as a receptor for its endogenous ligand 17{beta}-estradiol (E2) and is capable of binding a diverse set of natural and synthetic small molecules. Several screening campaigns have identified various ER ligands, based on the oxabicyclo[3.3.1]-nonene scaffold, that act as ER agonists. One such campaign extensively characterized an oxabicyclic ER agonist, termed AB-1. Herein, we report the synthesis of a series of analogs based on the scaffold of AB-1 (herein termed 1), focusing on the enhancement of their binding to ER and functional potencies in ER mediated assays. We identify a new substituted compound 9 that achieves subnanomolar potencies for ER binding (IC50= 0.45 nM) and ER dependent transcription (EC50= 0.37 nM) and proliferation (EC50= 0.03 nM), with potencies approaching those of E2. The high potency of 9 provides a promising scaffold for the potential development of new ER-targeted therapeutics.

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

The folate receptor (FR) is overexpressed in a range of human tumours including ovarian cancer cells. We propose that the overexpression of the FR on the surface of ovarian tumour cells could be exploited for the selective delivery of a DNA damage response inhibitor (DDRi) in the form of an intact folate drug conjugate (FDC). This approach would improve the therapeutic index of the parent DDRi facilitating combination studies of the DDRi-based FDC with DNA damaging chemotherapy. FR-mediated cellular uptake of the proposed folate drug conjugates is requisite for FDC selective delivery into tumours. In this study, we synthesised a series of olaparib-based folate conjugates that maintained the biochemical PARP1 inhibition associated with olaparib and showed binding affinity for the folate receptor. Significantly, we identified compounds 10b and 11 that selectively enter FR overexpressing tumour cells via folate receptor-mediated endocytosis in their intact form and engage with their target as demonstrated by the potent inhibition of PARylation (KB cells, PARylation IC50 = 5.7 and 3.9 nM; respectively).

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.