The amino-terminal segment in the β-domain of δ-cadinene synthase is essential for catalysis

Despite its distance from the active site the flexible amino-terminal segment (NTS) in the β-domain of the plant sesquiterpene cyclase δ-cadinene synthase (DCS) is essential for active site closure and desolvation events during catalysis

Effiziente chemoenzymatische Synthese von dhydroartemisinaldehyd

Artemisinin aus der Pflanze Artemisia annua ist das wirkungsvollste Arzneimittel zur Behandlung von Malaria. Die Sesquiterpen-Cyclase Amorphadien-Synthase, ein Cytochrom-abhängiges CYP450 und eine Aldehyd-Reduktase wandeln in der Pflanze Farnesyl-Diphosphat (FDP) in Dihydroartemisinaldehyd (DHAAl) um, welches ein Schlüsselzwischenprodukt in der Biosynthese von Artemisinin und eine halbsynthetische Vorstufe in der chemischen Synthese des Arzneimittels ist. Hier berichten wir über einen chemoenzymatischen Prozess, der in der Lage ist, DHAAl nur mithilfe der Sesquiterpen-Synthase aus einem gezielt synthetisierten, hydroxylierten FDP-Derivat herzustellen. Dieser Prozess, der die natürliche Sequenz aus Cyclisierung von FDP und Oxidation des Kohlenwasserstoffs umkehrt, stellt eine wesentliche Verbesserung der DHAAl-Synthese dar und zeigt das Potenzial neuer Substrate in der Terpen-Synthase-katalysierten Synthese hochwertiger Naturstoffe auf

Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae

The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography–mass spectrometry (GC–MS).  In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.  •We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs. •We validate the method by direct comparison with established assays. The agreement of kcat/KM among methods makes this adaptation optimal for rapid evaluation of TPSs. •We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries

Enzymatic synthesis of natural (+)-aristolochene from a non-natural substrate

The sesquiterpene cyclase aristolochene synthase from Penicillium roquefortii (PR-AS) has evolved to catalyse with high specificity (92%) the conversion of farnesyl diphosphate (FDP) to the bicyclic hydrocarbon (+)-aristolochene, the natural precursor of several fungal toxins. Here we report that PR-AS converts the unnatural FDP isomer 7-methylene farnesyl diphosphate to (+)-aristolochene via the intermediate 7-methylene germacrene A. Within the confined space of the enzyme's active site, PR-AS stabilises the reactive conformers of germacrene A and 7-methylene germacrene A, respectively, which are protonated by the same active site acid (most likely HOPPi) to yield the shared natural bicyclic intermediate eudesmane cation, from which (+)-aristolochene is then generated

Sulfided NiMo/<em>Clinoptilolite</em> Catalysts for Selective Sulfur Removal from Naphtha Stream without Olefin Hydrogenation

The natural clinoptilolite zeolite has been modified by acid leaching with HNO3 in order to obtain economic material for supporting NiMoS hydrotreating catalysts. The most optimized zeolite material was obtained by leaching with HNO3 at 80°C during 24 h. The bimetallic NiMo catalysts prepared by wet impregnation of a zeolite support, followed by calcination and sulfidation, were characterized by several physico-chemical techniques and tested in the hydrodesulfurization (HDS) of 3-methyl-thiophene (3-MT) model feed at atmospheric H2 pressure and T = 280°C. For all catalysts, the 3-MT transformation mainly occurs via direct desulfurization reaction route being diminished the catalyst hydrogenation function. This was linked with the formation of highly stacked layers of MoS2 particles having a low amount of “brim sites,” as demonstrated by HRTEM. The cause of the best performance of Ni-Mo(H)/Z-1 sulfide catalyst in the HDS of 3-MT can be the presence of K+ impurities on the support surface which forces the formation of highly stacked layers of MoS2 particles

Chemoenzymatic synthesis of the alarm pheromone (+)-verbenone from geranyl diphosphate

The enzyme-guided asymmetric synthesis of (+)-verbenone from geranyl diphosphate in a simple two-step, one pot transformation highlights the potential of chemoenzymatic procedures for the generation of high-value terpenoids

Reusable photocatalytic optical fibers for underground, deep-sea, and turbid water remediation

An approach for underground, deep, and turbid water remediation is presented based on optical fibers with a photocatalytic coating. Thus, photocatalytic TiO2 P25 nanoparticles immobilized in a poly(vinylidene difluoride) (PVDF) matrix are coated on polymeric optical fibers (POFs) and the photocatalytic performance of the system is assessed under artificial sunlight. To the best of our knowledge, poly(methyl methacrylate)-POF coated with TiO2/PVDF and the reusability of any type of POF for photocatalytic applications are not previously reported. The photocatalytic efficiency of the hybrid material in the degradation of ciprofloxacin (CIP) and its reusability are evaluated here. It is shown that 50 w/w% of TiO2 P25 achieves a degradation of 95% after 72 h under artificial sunlight and a reusability of three times leads to a loss of activity inferior to 11%. The efficient removal of ciprofloxacin and the stability of the POF coated with TiO2 P25 successfully demonstrate its suitability in the degradation of pollutants with potential application in regions with low light illumination, as in underground and deep water.Peer ReviewedPostprint (published version