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

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

Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-Germacrene A synthase

Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-3H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-2H6]-FDP support a germacrene–humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases

Structural Elucidation of Cisoid and Transoid Cyclization Pathways of a Sesquiterpene Synthase Using 2-Fluorofarnesyl Diphosphates

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (≥99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed

Emergence of terpene cyclization in Artemisia annua

The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-b-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of B27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism

Sequential degradation of organochlorinated herbicides by catalytic hydrodechlorination and photocatalysis

Trabajo presentado en el 3rd Latin-American Congress of Photocatalysis, Photochemistry and Photobiology, celebrado en San Luis Potosí (México) del 14 al 17 de octubre de 2014.In the last decade, there has been growing interest in development strategies more efficient to treat wastewater with organochlorinated compounds. The use of sequential or simultaneous hybrid configurations can improve both their detoxification and their final mineralization. One promising strategy is to couple an advanced catalytic process, as hydrodechlorination (HDC), with an advanced oxidation process, as heterogeneous photocatalysis. HDC undertakes an essential detoxification by means of transformation of organochlorinated compounds into their dechlorinated species, while photocatalysis can lead to complete mineralization to CO2 and H2O [2]. In this work, the combination of HDC and photocatalysis as an attractive sequential process to the degradation of chlorinated herbicides widely used in agriculture (MCPA, 2,4-D), has been evaluated. Both reactions were carried out in ambient conditions, using Pd/Al2O3 and P25 as catalysts, respectively. Ecotoxicity of the starting compounds and the reaction samples was determined by the Microtox Toxicity Test. Upon HDC, both chlorinated herbicides were completely degraded at 45 min reaction time, achieving an important decrease of the effluent ecotoxicity. However, upon photocatalysis, the original herbicides reached higher photodegradation rates, with more than 80 % of TOC removal, than the corresponding HDC effluent. Taking into account that the photo-efficiency of titania based-catalysts are usually related to a complex balance between their surface, textural, structural, morphological and electronic properties, the obtained results seem to indicate that the charge and the acidic/basic character of TiO2 surface is playing an important role on the final photodegradation of chlorinated herbicides at the acid pH values studied here.Wish to thank the Spanish MICINN and Comunidad de Madrid for their financial support for the projects CTQ 2010-14807, CTM 2010-14883 and S-2009/AMB-1588, respectively.Peer Reviewe

TiO2-reduced graphene oxide nanocomposites: Microsecond charge carrier kinetics

This Accepted Manuscript will be available for reuse under a CC BY-NC-ND licence after 24 months of embargo periodIn this work, transient absorption spectroscopy studies in the microsecond time scale were carried out to investigate the dynamics of photogenerated electron-hole pairs in TiO2-rGO nanocomposites, prepared by a hydrothermal method, under different atmospheres: N2, O2, and N2 saturated in CH3OH. Under N2 atmosphere, the transient absorption signal detected in the region between 450 and 700 nm dropped as the rGO mass concentration in the composite was raised. The electron transfer from TiO2 to rGO was confirmed by using a model based on fractal surfaces which describes the decay kinetics. In the presence of methanol as hole acceptor, P25-rGO 0.5% and 1% were able to reach the maximum transient absorption faster than the other studied nanocomposites. However, after 10 μs, the P25-rGO 0.1% nanocomposite yielded the highest transient absorption signal and the best conversion and initial reaction rate in the photocatalytic degradation of dichloroacetic acid in aqueous suspensions. The effect of rGO on free electrons was investigated by detecting the transient signal at 980 nm under N2 saturated in CH3OH, for the different samples. It was found that the measured signals followed the same response than at 660 nm further evincing the electron transfer process. No sensitization effect of rGO was observed when the samples were excited at 450 nm.This work has been supported by the Spanish Plan Nacional de I+D+i through the project CTM2015-64895-R. Alvaro Tolosana-Moranchel thanks to Ministerio de Educación, Cultura y Deporte for his FPU grant (FPU14/01605) and (EST16/00065). The authors are also grateful to Technical Research Support Unit of Instituto de Catálisis y Petroleoquímica (CSIC