RESUMEN
Vibralactone is isolated from the basidiomycete fungus Boreostereum vibrans as one of the strongest lipase inhibitors. Its unusual ß-lactone-fused bicycle is derived from an aryl ring moiety by an oxidative ring-expansion prior to an intramolecular cyclization. Herein, we report the discovery of the cyclase VibC which belongs to the α/ß-hydrolase superfamily and is involved in the vibralactone biosynthesis. Biochemical and crystal studies suggest that VibC may catalyze an aldol or an electrocyclic reaction initiated by the Ser-His-Asp catalytic triad. For the aldol and pericyclic chemistry in living cells, VibC is a unique hydrolase performing the carbocycle formation of an oxepinone to a fused bicyclic ß-lactone. This presents a naturally occurring, new enzymatic reaction in both aldol and hydrolase (bio)chemistry that will guide future exploitation of these enzymes in synthetic biology for chemical-diversity expansion of natural products.
Asunto(s)
Basidiomycota/química , Productos Biológicos/metabolismo , Hidrolasas/metabolismo , Lactonas/metabolismo , Biocatálisis , Productos Biológicos/química , Cristalografía por Rayos X , Ciclización , Hidrolasas/química , Lactonas/química , Lactonas/aislamiento & purificación , Modelos Moleculares , Estructura MolecularRESUMEN
A phytochemical investigation to obtain chemical components with potential anti-inflammatory activity from E. hylonoma led to the isolation of nine new ent-isopimarane diterpenoids (1 and 3-10), a new ent-rosane diterpenoid (11), along with eight known ones (2 and 12-18) using various chromatographic techniques. Compounds 3, 4, 5, and 10 were rare examples of the epoxy-ent-isopimarane. The structures of these new compounds were confirmed by extensive spectroscopic data, crystal X-ray diffraction analysis, and electronic circular dichroism. And the isolates were evaluated for their inhibitory effects on nitric oxide production induced by lipopolysaccharide in RAW 264.7 cells. The results showed that compounds 2 and 12 exhibited noteworthy inhibitory effects against NO production with IC50 values of 7.12 and 12.73⯵M, respectively, which were better than positive control (IC50â¯=â¯41.41⯵M). The possible mechanism that compounds 2 and 12 could inhibit NO production was investigated by the Western blotting experiments.
Asunto(s)
Diterpenos/química , Euphorbia/química , Animales , Antiinflamatorios , Macrófagos/efectos de los fármacos , Ratones , Modelos Moleculares , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Células RAW 264.7 , Relación Estructura-ActividadRESUMEN
Ten new triterpenoids, ailanaltiolides A-J (1-10), and three known analogues (11-13) were isolated from the roots of Ailanthus altissima. Compounds 1-7 are apotirucallane-type, compounds 8 and 9 are tirucallane-type, and compound 10 is a trinordammarane-type triterpenoid. This is the first study indicating the genus Ailanthus as a potential source for apotirucallane derivatives, which contain an α,ß-unsaturated-ε-lactone A-ring and diversely modified C-17 side chains. Spectroscopic data interpretation, electronic circular dichroism analysis, and X-ray crystallographic data defined the structures and absolute configurations of these triterpenoids. Compounds 2, 7, and 8 showed cytotoxicity against four tumor cell lines (HeLa, 786-O, HepG2, and A549). In particular, compound 2 exhibited the highest activity against 786-O cells with an IC50 value of 8.2 µM in vitro.
Asunto(s)
Ailanthus/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Raíces de Plantas/química , Triterpenos/química , Triterpenos/farmacología , Línea Celular Tumoral , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Difracción de Rayos XRESUMEN
The oxidative decarboxylation of prenyl 4-hydroxybenzoate to prenylhydroquinone has been frequently proposed for the biosynthesis of prenylated (hydro)quinone derivates (sometimes meroterpenoids), yet no corresponding genes or enzymes have so far been reported. A FAD-binding monooxygenase (VibMO1) was identified that converts prenyl 4-hydroxybenzoate into prenylhydroquinone and is likely involved in the biosynthesis of vibralactones and other meroterpenoids in the basidiomycete Boreostereum vibrans. Feeding of 3-allyl-4-hydroxybenzylalcohol, an analogue of the vibralactone pathway intermediate 3-prenyl-4-hydroxybenzylalcohol, generated 20 analogues with different scaffolds. This demonstrated divergent pathways to skeletally distinct compounds initiating from a single precursor, thus providing the first insight into a novel biosynthetic pathway for 3-substituted γ-butyrolactones from a shikimate origin.
Asunto(s)
Basidiomycota/enzimología , Vías Biosintéticas , Lactonas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Basidiomycota/química , Basidiomycota/metabolismo , Descarboxilación , Hidroquinonas/metabolismo , Lactonas/análisis , Parabenos/metabolismoRESUMEN
Oxepinone rings represent one of structurally unusual motifs of natural products and the biosynthesis of oxepinones is not fully understood. 1,5-Seco-vibralactone (3) features an oxepinone motif and is a stable metabolite isolated from mycelial cultures of the mushroom Boreostereum vibrans. Cyclization of 3 forms vibralactone (1) whose ß-lactone-fused bicyclic core originates from 4-hydroxybenzoate, yet it remains elusive how 4-hydroxybenzoate is converted to 3 especially for the oxepinone ring construction in the biosynthesis of 1. In this work, using activity-guided fractionation together with proteomic analyses, we identify an NADPH/FAD-dependent monooxygenase VibO as the key enzyme performing a crucial ring-expansive oxygenation on the phenol ring to generate the oxepin-2-one structure of 3. The crystal structure of VibO reveals that it forms a dimeric phenol hydroxylase-like architecture featured with a unique substrate-binding pocket adjacent to the bound FAD. Computational modeling and solution studies provide insight into the likely VibO active site geometry, and suggest possible involvement of a flavin-C4a-OO(H) intermediate.
Asunto(s)
Oxigenasas de Función Mixta , Proteómica , Lactonas/metabolismo , Flavinas , Flavina-Adenina DinucleótidoRESUMEN
Theaflavins (TFs) have attracted much attention due to their various bioactivities in black tea. This paper describes the first trial for enzymatic production of TFs by recombinant polyphenol oxidases (PPOs). PPO genes were cloned from nine species and expressed in E. coli. Crude enzyme assays by LC-MS revealed that eight recombinant PPOs were active for TFs production from tea polyphenols as substrates. Much higher activities were observed for crude enzymes of Md2 from Malus domestica (apple), Pp4 from Pyrus pashia (pear), and Ej2 from Eriobotrya japonica (loquat). When immobilized on mesoporous silica, crude Md2 was most active. The purified Md2 was immobilized and showed almost twice activity as high as its free enzyme. While the maximum activity of free enzyme was found at pHâ¯5 and 10-30⯰C, the immobilized enzyme had broader range of pHâ¯4-6 and 10-40⯰C. The activity of immobilized enzyme was relatively constant during the pH and thermal stability test. When used at 0.2â¯mg/ml in the beginning, the immobilized enzyme retained approximately 40% of its initial activity after 8â¯cycles of operation.
Asunto(s)
Biflavonoides/biosíntesis , Catequina/biosíntesis , Catecol Oxidasa/metabolismo , Polifenoles/metabolismo , Proteínas Recombinantes , Té/química , Biflavonoides/química , Catequina/química , Catecol Oxidasa/genética , Activación Enzimática , Estabilidad de Enzimas , Fermentación , Concentración de Iones de Hidrógeno , Estructura Molecular , Polifenoles/química , TermodinámicaRESUMEN
The Isodon plants (Lamiaceae) have been used in traditional Chinese medicine to alleviate sufferings from inflammations and cancers. This feature has been attributed to the presence of pharmacologically active ent-kaurane diterpenoids such as eriocalyxin B and oridonin. The Isodon eriocalyx (Dunn) Kudô species native to southwest China can accumulate a particularly high content of ent-kaurane diterpenoids (â¼1.5% w/w of dried leaves). We previously identified diterpene synthases IeCPS1 and IeCPS2 as ent-copalyl diphosphate synthases (ent-CPS) potentially involved in Isodon ent-kaurane diterpenoids biosynthesis. In this study, analysis of RNA-seq transcriptome of the I. eriocalyx plant revealed three other diterpene synthase genes (IeCPS3, IeKS1, and IeKSL1). Their functional characterization through coupled in vitro enzyme assays has confirmed that IeCPS3 is an ent-CPS specifically producing ent-copalyl diphosphate (ent-CPP). IeKS1 accepted ent-CPP to produce exclusively ent-kaurene and may thus be defined as an ent-kaurene synthase (ent-KS). When IeKSL1 was combined with IeCPS2 or IeCPS3, no product was detected. Based on tissue-specific expression and metabolic localization studies, the IeCPS3 and IeKS1 transcripts were significantly accumulated in leaves where the ent-kaurane diterpenoid eriocalyxin B dominates, whereas weak expression of both were observed in germinating seeds in which gibberellin biosynthetic pathway is normally active. Our findings suggest that both IeCPS3 and IeKS1 possess dual roles in general (gibberellins) and specialized diterpenoid metabolism, such as that of the Isodon ent-kaurane diterpenoids.