RESUMEN
Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize ß-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.
Asunto(s)
Deshidrogenasas-Reductasas de Cadena Corta , Stachybotrys , Oxidorreductasas , Catálisis , Alcoholes/químicaRESUMEN
Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Éteres Cíclicos , Paclitaxel , Sistema Enzimático del Citocromo P-450/metabolismo , Paclitaxel/biosíntesis , Paclitaxel/química , Paclitaxel/metabolismo , Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Taxus/enzimología , Taxus/metabolismo , Biocatálisis , Nicotiana/metabolismo , Nicotiana/enzimología , Estructura MolecularRESUMEN
Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue.
Asunto(s)
Benzopiranos , Flavonoides , Flavonoides/química , Glicosiltransferasas , PlantasRESUMEN
CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.
Asunto(s)
Diterpenos , Diterpenos/química , Diterpenos/metabolismo , Ciclización , Catálisis , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Sitios de UniónRESUMEN
Peridecalins C and D (1 and 2), one decalin and one oxygen-decalin containing polyketide-amino acid hybrids with 5/6/6 ring system, was isolated from a genetic mutant of Periconia sp. F-31. Their structures were elucidated through extensive spectroscopic data analysis, including 1 D/2D NMR and HR-MS spectra. Biosynthetically, two proposed Diels-Alder reactions are supposed to be involved in the skeleton construction of 1 and 2.
Asunto(s)
Ascomicetos , Policétidos , Aminoácidos , Ascomicetos/química , Estructura MolecularRESUMEN
One new eremophilane sesquiterpene periconianone L (1), together with four known guaiane-type sesquiterpenes 4,10,11-trihydroxyguaiane (2), (-)-guai-1(10)-ene-4α,11-diolhydroxymecuration (3), guaidiol A (4), and epi-guaidiol A (5) were isolated from the endophytic fungus Periconia sp. F-31. The structure of the new compound was established by spectroscopic methods, including UV, IR, HRESIMS, and extensive NMR techniques. Compound 3 was isolated as natural product for the first time.
Asunto(s)
Ascomicetos , Sesquiterpenos , Ascomicetos/química , Estructura Molecular , Sesquiterpenos Policíclicos , Sesquiterpenos/químicaRESUMEN
Various bioactive polyketides have been found in Aloe barbadensis. However, the polyketide synthases (PKSs), which participate in biosynthesis of polyketides in A. barbadensis remain unknown. In this study, two type III PKSs (AbPKS1 and AbPKS2) were identified from A. barbadensis. AbPKS1 and AbPKS2 were able to utilize malonyl-CoA to yield heptaketides (TW93a and aloesone) and octaketides (SEK4 and SEK4b), respectively. AbPKS1 also exhibited catalytic promiscuity in recognizing CoA thioesters of aromatics to produce unusual polyketides. What Is more, a whole cell biocatalysis system with the capability of producing 26.4 mg/L of SEK4/SEK4b and 2.1 mg/L of aloesone was successfully established.
Asunto(s)
Aloe , Policétidos , Aciltransferasas , Estructura Molecular , Sintasas PoliquetidasRESUMEN
Bistachybotrysin K (1), one new phenylspirodrimane dimer with a central 6/7 oxygen heterocycle core, was isolated from the fungus Stachybotrys chartarum CGMCC 3.5365. Its structure was elucidated by extensive spectroscopic data and single-crystal X-ray diffraction. Compound 1 showed significant cytotoxicity against human tumor cell lines HCT116, NCI-H460, BGC823, Daoy, and HepG2 with IC50 values in the range of 1.1-4.7 µM.
Asunto(s)
Antineoplásicos , Compuestos de Espiro , Stachybotrys , Línea Celular Tumoral , Humanos , Estructura MolecularRESUMEN
Icariin (1a), a 7-O-glycosylated flavonoid glycoside, is recognized as the major pharmacologically active ingredient of Epimedium plants, which have been used in traditional Chinese medicine for thousands of years. However, no glycosyltransferase (GT) responsible for the 7-O-glycosylation of flavonoids has been identified from Epimedium plants to date. Herein, a GT, Ep7GT, was identified from E. pseudowushanense B. L. Guo, which can regiospecifically transfer a glucose moiety to baohuoside (1) at 7-OH to form icariin (1a). Ep7GT showed a rare broad donor substrate spectrum, including UDP-glucose, UDP-xylose, UDP-N-acetylglucosamine, UDP-rhamnose, UDP-galactose, UDP-glucuronic acid and TDP-glucose. Moreover, two new derivatives of icariin (1a), 7-O-ß-d-[2-(acetylamino)-2-deoxy-glucopyranosyl]-baohuoside (1b) and 7-O-ß-d-xylosyl-baohuoside (1c), were biosynthesized by using Ep7GT in vitro. Engineered Escherichia coli harbouring Ep7GT was constructed, and 10.1 µg mL-1 icariin (1a) was yielded by whole-cell biotransformation with baohuoside (1) as the substrate. The present work not only characterizes the GT responsible for the 7-O-glycosylation in the biosynthesis of icariin in Epimedium plants, but also indicates the significant potential of an enzymatic approach for the production of glycosylated baohuoside derivatives with different sugar moieties. What's more, these findings also provide a promising alternative for producing natural/unnatural bioactive flavonoid glycosides by metabolic engineering.
Asunto(s)
Epimedium/enzimología , Flavonoides/biosíntesis , Glicosiltransferasas/metabolismo , Azúcares/metabolismo , Biocatálisis , Biotransformación , Flavonoides/química , Glicosilación , Glicosiltransferasas/química , Conformación Molecular , Azúcares/químicaRESUMEN
Three new phenylspirodrimanes derivatives named stachybotrysins H and I (1 and 2) and stachybotrin E (3), together with one known compound stachybotrylactam (4), were isolated from Stachybotrys chartarum CGMCC 3.5365. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 1 and 2 showed inhibitory effect towards potassium channel Kv1.3 with IC50 values of 13.4 and 10.9 µM, respectively.
Asunto(s)
Canal de Potasio Kv1.3/antagonistas & inhibidores , Compuestos de Espiro/química , Stachybotrys/química , Animales , Células CHO , Línea Celular , Cricetinae , CricetulusRESUMEN
Bistachybotrysins A-C (1-3), three phenylspirodrimane dimers representing an unusual [6,6,7,6]-tetracyclic skeleton with a central 2,10-dioxabicyclo[4.3.1]decan-7-ol core fused with two phenyl units, were isolated from a fungal strain, Stachybotrys chartarum CGMCC 3.5365. The structures of 1-3 were elucidated through extensive spectroscopic data analysis, including Mo2(AcO)4-induced and calculated electronic circular dichroism (ECD). 1 and 2 exhibited potent cytotoxicity against four human tumor cell lines with IC50 values in the range of 2.8-7.5⯵M. Furthermore, a possible biogenesis for 1-3 is proposed.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Compuestos de Espiro/farmacología , Stachybotrys/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Compuestos de Espiro/química , Compuestos de Espiro/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Epimedium is used in traditional Chinese medicine and contains flavonol glycosides that exhibit multiple biological activities. These bioactive flavonol glycosides usually have a rhamnose moiety at the 3-OH position of prenylflavonols, such as icariin (9), baohuoside I (1a) and baohuoside II (2a). However, to date, no rhamnosyltransferase has been reported to catalyze the 3-O-rhamnosylation of prenylflavonols. In this article, a flavonol rhamnosyltransferase, EpPF3RT, was identified from E. pseudowushanense B. L. Guo. The recombinant enzyme regiospecifically transfers a rhamnose moiety to 8-prenylkaempferol (1) and anhydroicaritin (2) at the 3-OH position to form baohuoside II (1a) and baohuoside I (2a) in vitro. In addition, a UDP-rhamnose synthase gene, EpRhS, from E. pseudowushanense was functionally characterized and used to produce the UDP-rhamnose sugar donor. Furthermore, an engineered Escherichia coli strain containing EpPF3RT and EpRhS was established to produce baohuoside II (1a) from whole cells. These studies indicate the significant potential of an enzymatic approach for the rhamnosylation of bioactive flavonoids in Epimedium plants and will provide a promising alternative for producing bioactive rhamnosylated flavonoids combined with other genes/enzymes by synthetic biology.
Asunto(s)
Biocatálisis , Epimedium/enzimología , Flavonoles/química , Flavonoles/metabolismo , Hexosiltransferasas/metabolismo , Ramnosa/metabolismo , Flavonoides/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
Glucuronidation is an important and popular metabolic reaction in vivo of drugs. The further evaluation of biological activity and toxicity of glucuronides is necessary in the course of the drug research and development. However, the synthesis of glucuronides is limited by the lack of efficient approach. Herein, we have developed a new glucuronide synthesis method using plant uridine diphosphate-dependent glucuronosyltransferases (UGTs), UGT88D4, UGT88D7, and EpGT8, enabling the convenient preparation for corresponding O-glucuronide metabolites (1a, 2a, 3a, and 3b) in milligram scale of two neurological active agents, IMM-H004 (1) and FLZ (2). Their structures were characterized by spectroscopic data analyses.
Asunto(s)
Glucurónidos/síntesis química , Glucuronosiltransferasa/metabolismo , Plantas/enzimología , Clonación Molecular , Glucuronosiltransferasa/química , Glucuronosiltransferasa/genética , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Metales/química , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
Two new lanostane triterpenoids (1 and 2), two new ergostane-type steroids (3 and 4) together with two known lanostane triterpenoids (5 and 6) and one known steroid (7) were isolated from the cultured mycelia of Ganoderma capense (CGMCC 5.71). Their structures were determined on the basis of extensive spectroscopic (HRESIMS, 1D NMR, 2D NMR) data analyses. Compound 1 exhibited moderate cytotoxic activity against the human cancer cell line NCI-H1650 with an IC50 value of 22.3 µM, and 7 displayed cytotoxic activity against the human cancer cell line HCT116 with an IC50 value of 17.4 µM. In addition, compounds 2, 3, 5, and 6 displayed weak anti-HIV activity with IC50 values of 23.5, 46.7, 21.6, and 30.1 µM, respectively.
Asunto(s)
Ganoderma/química , Micelio/química , Esteroides/química , Triterpenos/química , Ganoderma/metabolismo , Estructura Molecular , Micelio/metabolismo , Esteroides/metabolismoRESUMEN
In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level.IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.
Asunto(s)
Absidia/enzimología , Glicósidos/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Fenoles/metabolismo , Rhizopus/enzimología , Uridina Difosfato/metabolismo , Absidia/genética , Absidia/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilación , Glicosiltransferasas/química , Glicosiltransferasas/aislamiento & purificación , Familia de Multigenes , Filogenia , Rhizopus/genética , Rhizopus/metabolismoRESUMEN
Seven new phenylspirodrimane derivatives named stachybotrysins A-G (2-8), together with five known compounds (1, 9-12), were isolated from Stachybotrys chartarum CGMCC 3.5365. Stachybotrysin D (5) is the first reported example of a naturally occurring alcoholic O-sulfation of a phenylspirodrimane, and stachybotrysins F and G (7 and 8) are the first examples possessing an isobenzotetrahydrofuran ring with an acetonyl moiety attached. The structures of these compounds were elucidated on the basis of extensive spectroscopic data analysis and by comparison with reported data. The absolute configurations of 1-8 were determined by X-ray single-crystal diffraction, electronic circular dichroism (ECD), and calculated ECD. Compounds 1 and 8 displayed anti-HIV activity with IC50 values of 15.6 and 18.1 µM, respectively, and 2, 7, 9, and 11 showed inhibitory effect on influenza A virus with IC50 values ranging from 12.4 to 18.9 µM.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Antivirales/aislamiento & purificación , Sesquiterpenos/aislamiento & purificación , Compuestos de Espiro/aislamiento & purificación , Stachybotrys/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Antivirales/química , Antivirales/farmacología , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Virus de la Influenza A/efectos de los fármacos , Concentración 50 Inhibidora , Conformación Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Sesquiterpenos/farmacología , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
A new steroid glucoside (1), along with nine known steroids (2-10) and four known sorbicillinoids (11-14), were isolated from the endophytic fungus Trichoderma sp. Xy24. Their structures were elucidated on the basis of spectroscopic data analyses and by comparison with reported data. Compounds 3, 5-7, 9, 10, and 13 exhibited significant inhibitory effects on HIV-1 virus with IC50 values ranging 1.9-9.3 µM; compounds 10, 13, and 14 showed potent inhibitory activity on LPS-induced NO production in BV2 microglia cells with inhibitory rates of 108.2, 100, and 75.1% at 10 µM, respectively. In addition, compound 10 displayed moderate cytotoxicity against BCG823 and HePG2 cell lines with IC50 values of 11.1 and 17.7 µM, respectively.
Asunto(s)
Glucósidos/aislamiento & purificación , Glucósidos/farmacología , Esteroides/aislamiento & purificación , Esteroides/farmacología , Trichoderma/química , Antiinflamatorios no Esteroideos/farmacología , Glucósidos/química , Células HCT116 , VIH-1/efectos de los fármacos , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Microglía/efectos de los fármacos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Paclitaxel/farmacología , Esteroides/químicaRESUMEN
Three new sesquiterpenoids trichoacorenols B-C and cyclonerodiol B (1-3), along with three known ones (4-6), were isolated from the mangrove plant endophytic fungus Trichoderma sp. Xy24 using various column chromatography techniques. The structures of these compounds were determined on the basis of extensive spectroscopic data analyses. Compounds 1, 2, 4, and 5 were four acorane sesquiterpenes, 3 and 6 were two monocyclic sesquiterpenediols. Compounds 3 and 5 exhibited significant neural anti-inflammatory activity by inhibiting LPS-induced NO production in BV2 cells with the inhibitory rates of 75.0% and 39.2% at 0.1 µM, respectively, which are more potent than curcumin, a positive control with the inhibitory rate of 21.1% at 0.1 µM.
Asunto(s)
Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Trichoderma/química , Animales , Antiinflamatorios/química , Curcumina/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Estructura Molecular , Óxido Nítrico/biosíntesis , Rhizophoraceae/microbiología , Sesquiterpenos/químicaRESUMEN
Five monoterpenoids were isolated from the endophytic fungus Periconia sp. F-31, including three new carene-type monoterpenoids, 2-carene-5,8-diol (1), 2-carene-8,10-diol (2), 2-carene-8-acetamide (3), one new menthene-type monoterpenoid 8-hydroxy-1,7-expoxy-2-menthene (4), and one known monoterpenoid anethofuran (5). The structures of all compounds were elucidated based on a comprehensive spectroscopic data analysis, electronic circular dichroism (ECD), and calculated ECD.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Ascomicetos/química , Monoterpenos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/farmacología , Monoterpenos Bicíclicos , Dicroismo Circular , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Monoterpenos/química , Monoterpenos/farmacología , Resonancia Magnética Nuclear BiomolecularRESUMEN
The glycosides of 4'-demethylepipodophyllotoxin (DMEP) possess various pharmacological activities; however, the chemical synthesis of these glycosides faces challenges in regioselectivity, stereoselectivity, and the protection and de-protection of functional groups. In this work, a novel glycosyltransferase (GT) gene AbGT5 from Aloe barbadensis was successfully cloned, heterogeneously expressed and purified. Recombinant AbGT5 was able to catalyze the glycosylation of DMEP and the glycosylated product, which was separated from the preparative scale reaction, was characterized as DMEP 4'-O-ß-D-glucoside via MS, 1H-NMR, 13C-NMR, HSQC and HMBC. According to the investigations of enzyme properties, AbGT5 show the highest activity around 20 â in the buffer of pH 9.0, and it was independent of divalent metal ions. Under the optimum conditions, the conversion rate of DMEP can reach 80%. Above all, in this work the enzymatic glycosylation of DMEP was achieved with high efficiency by the novel GT AbGT5.