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

An efficient chemoenzymatic synthesis of dihydroartemisinic aldehyde

Artemisinin from the plant Artemisia annua is the most potent pharmaceutical for the treatment of malaria. In the plant, the sesquiterpene cyclase amorphadiene synthase, a cytochrome- dependent CYP450, and an aldehyde reductase convert farnesyl diphosphate (FDP) into dihydroartemisinic aldehyde (DHAAl), which is a key intermediate in the biosynthesis of artemisinin and a semisynthetic precursor for its chemical synthesis. Here, we report a chemoenzymatic process that is able to deliver DHAAl using only the sesquiterpene synthase from a carefully designed hydroxylated FDP derivative. This process, which reverses the natural order of cyclization of FDP and oxidation of the sesquiterpene hydrocarbon, provides a significant improvement in the synthesis of DHAAl and demonstrates the potential of substrate engineering in the terpene synthase mediated synthesis of highvalue natural products

TiO2 and TiO2-SiO2 coated cement: Comparison of mechanic and photocatalytic properties

peer reviewedAn increasingly known application of nanomaterials in the construction industry is related to the photoactivity ability of semiconductors, where nano-anatase TiO2 is perhaps the most well-known photocatalytic semiconductor and one which possesses a strong oxidizing capability. To analyze the photo-efficiency of four TiO2 coatings and the effect of SiO2 interlayer on the mechanic and photocatalytic activity in Rhodamine B (RhB) and NOx photodegradation, two photocatalytic cement series have been prepared. First, cement mortar was coated with three commercial TiO2 suspensions (GG1, GC7 and CG13) and a home-made titania sol-gel (TEA), and secondly an insertion of a SiO2 layer was applied on cement surface before spraying the TiO2 layer.All studied TiO2-cements exhibited a significant RhB and NOx photodegradation, arising almost total RhB molar conversions, and upper 53% for NOx photo-oxidation respectively. Nevertheless, although SiO2 layer deposited in between mortar and TiO2-cement did not stabilize the commercial TiO2 coatings, a good adhesion was observed when silica was applied joint to the home-made titania gel (TEA), probably as a consequence of the interactions encountered between SiO2 and TiO2 gels. CG7-Si-Cem exhibited high rate at shorter irradiation times, but TEA-Cem and TEA-Si-Cem can be considered as very interesting and potential photocatalytic mortars due to useful mechanical properties, with a very good coatings adhesion that provides promising outdoor use, and good photo-efficiencies in RhB and NOx photo-oxidation

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