Labeling Experiments Research Articles

Solar-driven nitrogen fixation with bio-inspired Mo/Sr-MOFs photocatalysts at ambient conditions

Photocatalytic reduction of nitrogen to ammonia has attracted much attention recently because ammonia stands out as one of the indispensable chemicals on a global scale. Herein, bio-inspired MOFs Mo-NTC and Sr-NTC were designed and synthesized by using Mo or Sr as metal nodes and 1,4,5,8-naphthalenetetracarboxylic acid (H4NTC) as ligands which has visible-light responsiveness. The best photocatalytic nitrogen fixation performance of Sr-NTC was about 143.10 μmol·g−1·h−1 which is higher than Mo-NTC (70.20 μmol·g−1·h−1). The ability of Sr-NTC to convert nitrogen to ammonia was verified by 15N2 isotope labeling experiment and in-situ FTIR measurement. Various characterization methods were employed to confirm the enhanced performance of photocatalytic nitrogen reduction. This work provides a new idea for applying metal Sr and Mo and organic ligand to construct photocatalysis to fix nitrogen driven by solar at ambient conditions.

Elucidating the mechanism of the FeCl3-Catalyzed quinoline synthesis by A3 multicomponent reaction

Access to the quinoline scaffold through a multicomponent A3 (Aldehyde-Amine-Alkyne) protocol has emerged as a successful synthetic route owing to their experimental simplicity and relevant sustainable potential. Since the original report for A3 synthesis of quinolines based on Cu(I) catalyst, a substantial part of scientific literature has considered propargylamine formation via acetylide metal coupling as the key reaction intermediate for many Lewis acid catalysts, despite the obvious competition with a Povarov-like [4 + 2] cycloaddition mechanism. Considering the positive economic and environmental impacts of using non-precious metal catalysts, and the lack of solid evidence to support the preferential pathway in Fe(III)-catalyzed quinoline synthesis, we employed isotopic labeling and kinetic experiments associated with DFT calculations to support the formal [4 + 2] cycloaddition as the most favorable mechanism in A3-Fe catalyzed MCR protocol under aerobic conditions.

BayFlux: A Bayesian method to quantify metabolic Fluxes and their uncertainty at the genome scale.

Metabolic fluxes, the number of metabolites traversing each biochemical reaction in a cell per unit time, are crucial for assessing and understanding cell function. 13C Metabolic Flux Analysis (13C MFA) is considered to be the gold standard for measuring metabolic fluxes. 13C MFA typically works by leveraging extracellular exchange fluxes as well as data from 13C labeling experiments to calculate the flux profile which best fit the data for a small, central carbon, metabolic model. However, the nonlinear nature of the 13C MFA fitting procedure means that several flux profiles fit the experimental data within the experimental error, and traditional optimization methods offer only a partial or skewed picture, especially in "non-gaussian" situations where multiple very distinct flux regions fit the data equally well. Here, we present a method for flux space sampling through Bayesian inference (BayFlux), that identifies the full distribution of fluxes compatible with experimental data for a comprehensive genome-scale model. This Bayesian approach allows us to accurately quantify uncertainty in calculated fluxes. We also find that, surprisingly, the genome-scale model of metabolism produces narrower flux distributions (reduced uncertainty) than the small core metabolic models traditionally used in 13C MFA. The different results for some reactions when using genome-scale models vs core metabolic models advise caution in assuming strong inferences from 13C MFA since the results may depend significantly on the completeness of the model used. Based on BayFlux, we developed and evaluated novel methods (P-13C MOMA and P-13C ROOM) to predict the biological results of a gene knockout, that improve on the traditional MOMA and ROOM methods by quantifying prediction uncertainty.

Heteroditopic NHC Ligand Supported Manganese(I) Complexes: Synthesis, Characterization, and Activity as Non-bifunctional Phosphine-Free Catalyst for the α-Alkylation of Nitriles.

In the present work, several manganese(I) complexes of chelating heteroditopic ligands Mn1-3, featuring ImNHC (imidazol-2-ylidene) connected to a 1,2,3-triazole-N or tzNHC (1,2,3-triazol-5-ylidene) donors via a methylene spacer, with possible modifications at the triazole backbone have been synthesized and completely characterized. Notably, the CO stretching frequencies, electrochemical analysis, and frontier orbital analysis certainly suggest that the chelating ImNHC-tzNHC ligands have stronger donation capabilities than the related ImNHC-Ntz ligand in the synthesized complexes. Moreover, these well-defined phosphine-free Mn(I)-NHC complexes have been found to be effective non-bifunctional catalysts for the α-alkylation of nitriles using alcohols and importantly, the catalyst Mn1 containing ImNHC connected to a weaker triazole-N donor displayed higher activity compared to Mn2/Mn3 containing an unsymmetrical bis-carbene donors (ImNHC and tzNHC). A wide range of aryl nitriles were coupled with diverse (hetero)aromatic as well as aliphatic alcohols to get the corresponding products in good to excellent yields (32 examples, up to 95 % yield). The detailed mechanistic studies including deuterium labelling experiments reveal that the reaction follows a Borrowing Hydrogen pathway.

Elucidating the functional role of human ABHD16B lipase in regulating triacylglycerol mobilization and membrane lipid synthesis in Saccharomyces cerevisiae

Lipids are essential biological macromolecules that play a pivotal role in various physiological processes and cellular homeostasis. ABHD16B, a member of the α/β-hydrolase domain (ABHD) superfamily protein, has emerged as a potential key regulator in lipid metabolism. However, the precise role of human ABHD16B in lipid metabolism remains unclear. In this study, we reported the overexpression of ABHD16B in Saccharomyces cerevisiae to determine its physiological relevance in lipid metabolism. Through in vivo [14C]acetate labeling experiments, we observed that overexpression of ABHD16B causes a decrease in cellular triacylglycerol (TAG) levels and a concurrent increase in phospholipid synthesis in wild-type cells. Mass spectrometry (LC–MS/MS) analysis further corroborated these findings, showing a significant decrease in TAGs with a carbon chain length of 48 and an increase in major phospholipid species, specifically 34:2, upon overexpression of ABHD16B. Confocal microscopy analysis revealed a reduction in the number of lipid droplets in strains overexpressing ABHD16B, consistent with the observed decrease in neutral lipids. Additionally, qRT-PCR analysis indicated a high phospholipid synthetic activity of ABHD16B and a potential decrease in TAG levels in wild-type yeast, possibly due to upregulation of endogenous TAG hydrolytic enzymes, as confirmed using 3tglsΔ mutant strain. Furthermore, GC-MS analysis revealed significant modifications in fatty acid composition upon ABHD16B overexpression. Collectively, our results underscore the influence of ABHD16B overexpression on TAG levels, phospholipid synthesis, lipid droplet dynamics, and fatty acid composition. These findings reveal a complex interplay between TAG hydrolysis and phospholipid synthesis, highlighting the critical involvement of ABHD16B in lipid homeostasis and providing further insights into its regulatory function in cellular lipid metabolism.

Structural characterization of a 3-hydroxychromone dye trehalose conjugate for fluorescent labelling of mycobacteria

3-Hydroxychromone (3HC) dye trehalose conjugates have previously been exploited as environment-sensitive fluorescent labels to detect mycobacteria by fluorescence microscopy. We have studied the 3HC dye 2-(6-(diethylamino)benzofuran-2-yl)-3-hydroxy-4H-chromen-4-one (3HC-2) trehalose conjugate 3HC-2-Tre by X-ray crystallography, spectroscopic methods augmented by DFT calculations and conducted preliminary labelling experiments with Mycobacterium abscessus, a non-tuberculous mycobacterium and opportunistic pathogen. In the crystal structure of 3HC-2-Tre ⋅ 3.14 H2O, the 3HC-2-Tre molecules exhibit an intramolecular OH⋅⋅⋅O hydrogen bond between the 5-hydroxy group of trehalose and the chromone carbonyl oxygen atom, and form hydrogen-bonded dimers via the trehalose units with approximate local C2 symmetry. The chromophore adopts an s-cis conformation of the chromone and benzofuran systems about its central CC bond, as previously encountered in the crystal structure of the free dye 3HC-2, and the trehalose moieties approximately retain the shape as found in the crystal structures of anhydrous trehalose and the dihydrate. Absorption spectra of 3HC-2-Tre acquired in four different solvents revealed small positive solvatochromy. Fluorescence microscopy detected M. abscessus cells treated with 3HC-2-Tre in the GFP channel.

Coupled carbon and nitrogen cycling regulates the cnidarian–algal symbiosis

Efficient nutrient recycling underpins the ecological success of cnidarian-algal symbioses in oligotrophic waters. In these symbioses, nitrogen limitation restricts the growth of algal endosymbionts in hospite and stimulates their release of photosynthates to the cnidarian host. However, the mechanisms controlling nitrogen availability and their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Combining 13C and 15N stable isotope labeling experiments with physiological analyses and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by labile carbon availability. Light regimes optimal for algal photosynthesis increase carbon availability in the holobiont and stimulate nitrogen assimilation in the host metabolism. Consequently, algal symbiont densities are lowest under optimal environmental conditions and increase toward the lower and upper light tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. Yet, the dependence on resource competition may favor parasitic interactions, explaining the instability of the cnidarian-algal symbiosis as environmental conditions in the Anthropocene shift towards its tolerance limits.

Assessing the allocations of exogenous N to the soil organic N pool in maize–wheat cropping using 15N in situ labelling

The accumulation of nitrogen (N) from straw and fertilizer in soil effectively reduces N losses, which is vital for protecting dryland farming environments. However, the quantification of exogenous N contributions to soil organic nitrogen (SON) under different carbon (C) and N management practices in maize–wheat cropping systems remains unknown. Here, a 15N in situ labelling experiment was conducted, based on continuous 5-year N levels (0, 150, 250 kg N ha−1 applied for each crop) and two straw management practices (NS, straw removal; AS, straw incorporation) to investigate the allocation of exogenous N to SON and its underlying accumulation mechanisms. The atom% excess in SON was determined after fractionating it into active and stable fractions by the acid hydrolysis method. Compared to NS, AS significantly increased the distribution of fertilizer N into stable SON by 168.4 %–223.6 % in the maize season, and into active and stable SON by 256.7 %–278.4 % and 142.0 %–167.6 %, respectively, in the wheat season. The content and retention rate of fertilizer N in SON were highest at the N250 and N150 levels, respectively, under both NS and AS treatments in the two crop seasons. In contrast, N addition decreased the allocation of straw N to SON, especially in the wheat season. Notably, the content and residual rate of exogenous N in SON between the N150 and N250 levels showed no significant differences. Straw incorporation exerted the most significant direct and positive impact on the immobilization of fertilizer N in the soil, whereas N application indirectly influenced straw N accumulation, primarily by altering labile C and N contents, subsequently selecting specific microbial communities. Gram-positive bacteria and actinomycetes exhibited a significant positive correlation with straw N content in SON. This study provides a new perspective on N nutrient management by quantifying exogenous N accumulation in the soil.

Electron ionization fragmentation studies for a series of 4-methoxymethylene benzoate esters.

Product ion studies and stable isotope deuterium labeling experiments provide useful data for understanding the electron ionization (EI)-mass spectroscopy (MS) fragmentation of methoxymethylene substituted benzoate esters. The methoxymethylene ether is regioisomeric with the ethoxy group and represents the two possible ether substituents of a benzene ring of C2 H5 O. Structural confirmation of these synthetic precursor materials via gas chromatography (GC)-EI-MS revealed unexpected fragment ions. The synthesis and EI-MS evaluation of some homologs and deuterated derivatives allowed for the characterization of these unique ions and their fragmentation pathways. The relative effects of the position of the oxygen of the ether side chain are the subject of this investigation. The desired compounds were prepared from 4-chloromethylbenzoyl chloride by alkoxide displacement followed by transesterifications and the deuterated analogs were prepared similarly. The compounds were separated by capillary GC and their MS fragmentation evaluated in EI, MS/MS and chemical ionization experiments. The methoxymethylene-substituted benzoate esters yield major fragment ions from the loss of the alkyl group from the ether as well as alkoxy group loss from the ester or ether portion of the molecule. The loss of the alkyl group from the ether followed by loss of the ester alkoxy group as the corresponding alcohol yielded the unique cation at m/z 133 for all compounds. The identity of the major ions was confirmed by product ion and deuterium labeling studies and possible mechanisms of fragment ion formation are described. The aliphatic oxygen of the alkoxymethylene group plays a much more active role in the EI-MS fragment formation profile than the direct aromatic ring linked oxygen of the ethoxy group. Thus, yielding a greater variety of characteristic fragments. The m/z 133 ion is unique to this class of compounds and does not have an equivalent pathway for the regioisomeric ethoxy series.

Repeated mild traumatic brain injury causes sex-specific increases in cell proliferation and inflammation in juvenile rats

Childhood represents a period of significant growth and maturation for the brain, and is also associated with a heightened risk for mild traumatic brain injuries (mTBI). There is also concern that repeated-mTBI (r-mTBI) may have a long-term impact on developmental trajectories. Using an awake closed head injury (ACHI) model, that uses rapid head acceleration to induce a mTBI, we investigated the acute effects of repeated-mTBI (r-mTBI) on neurological function and cellular proliferation in juvenile male and female Long-Evans rats. We found that r-mTBI did not lead to cumulative neurological deficits with the model. R-mTBI animals exhibited an increase in BrdU + (bromodeoxyuridine positive) cells in the dentate gyrus (DG), and that this increase was more robust in male animals. This increase was not sustained, and cell proliferation returning to normal by PID3. A greater increase in BrdU + cells was observed in the dorsal DG in both male and female r-mTBI animals at PID1. Using Ki-67 expression as an endogenous marker of cellular proliferation, a robust proliferative response following r-mTBI was observed in male animals at PID1 that persisted until PID3, and was not constrained to the DG alone. Triple labeling experiments (Iba1+, GFAP+, Brdu+) revealed that a high proportion of these proliferating cells were microglia/macrophages, indicating there was a heightened inflammatory response. Overall, these findings suggest that rapid head acceleration with the ACHI model produces an mTBI, but that the acute neurological deficits do not increase in severity with repeated administration. R-mTBI transiently increases cellular proliferation in the hippocampus, particularly in male animals, and the pattern of cell proliferation suggests that this represents a neuroinflammatory response that is focused around the mid-brain rather than peripheral cortical regions. These results add to growing literature indicating sex differences in proliferative and inflammatory responses between females and males. Targeting proliferation as a therapeutic avenue may help reduce the short term impact of r-mTBI, but there may be sex-specific considerations.

Subrutilan – Ein Hexacyclisches Sesterterpen aus Streptomyces subrutilus

AbstractDie Untersuchung einer Terpensynthase aus Streptomyces subrutilus führte zur Identifizierung des hexacyclischen Sesterterpens Subrutilan sowie von acht pentacyclischen Nebenprodukten. Subrutilan stellt den ersten Fall eines gesättigten Sesterterpenkohlenwasserstoffs dar. Seine Struktur einschließlich der absoluten Konfiguration wurde durch Röntgenstrukturanalyse und stereoselektive Deuterierung eindeutig bestimmt. Der Cyclisierungsmechanismus zu Subrutilan und seinen Nebenprodukten wurde durch Isotopenmarkierungsexperimente und DFT‐Rechnungen detailliert untersucht. Die Subrutilan‐Synthase (SrS) wandelte auch (2Z)‐GFPP in ein Hauptprodukt um. Weitere Verbindungen wurden aus den Substratanaloga (7R)‐6,7‐Dihydro‐GFPP und (2Z,7R)‐6,7‐Dihydro‐GFPP mit blockierter Reaktivität in der C6−C7‐Bindung erhalten. Interessanterweise verlaufen die frühen Schritte der Cyclisierungskaskade mit (2Z)‐GFPP und den gesättigten Substratanaloga analog zu denen von GFPP, doch dann kommt es zu Abweichungen vom natürlichen Cyclisierungsmodus.

The Evolution of Hexagonal Cobalt Nanosheets for CO2 Electrochemical Reduction Reaction

The CO2 electrochemical reduction reaction (CO2RR) is one of the most promising methods to reduce carbon dioxide emissions and store energy. At the same time, the pathways of CO2 reduction reaction are diverse and the products are abundant. Converting carbon dioxide to C2+ products, a critical feedstock, requires a C–C coupling step with the transfer of more than 10 electrons per molecule and, hence, is kinetically sluggish. The production of some key adsorptions is conducive to the formation of C2+ products. In this work, we used in situ techniques to figure out the reason why hexagonal-close-packed (hcp) Co nanosheets (NSs) have high activity in CO2RR to ethanal. According to the in situ Raman spectra, the high local pH environment on the catalyst surface is favorable for CO2RR. The high pH at low potentials not only suppresses the competing hydrogen evolution reaction but also stimulates the production of COCO* intermediate. The isotopic labeling experiment in differential electrochemical mass spectrometry (DEMS) provides a possible sequence of the products. The 13CO is generated when we replace 12CO2 with 13CO2, which identifies the origin of the products. Besides, in situ electrochemical impedance spectroscopy (EIS) shows that the hcp Co at −0.4 V vs. RHE boosts the H2O dissociation and proton transfer, feeding sufficient H* for CO2 to *COOH. In the end, by analyzing the transmission electronic microscopy (TEM), we find that the Co (002) plane may be beneficial to the conversion of CO2 and the adsorption of intermediates.

Highly active, ultra-low loading single-atom iron catalysts for catalytic transfer hydrogenation

Highly effective and selective noble metal-free catalysts attract significant attention. Here, a single-atom iron catalyst is fabricated by saturated adsorption of trace iron onto zeolitic imidazolate framework-8 (ZIF-8) followed by pyrolysis. Its performance toward catalytic transfer hydrogenation of furfural is comparable to state-of-the-art catalysts and up to four orders higher than other Fe catalysts. Isotopic labeling experiments demonstrate an intermolecular hydride transfer mechanism. First principles simulations, spectroscopic calculations and experiments, and kinetic correlations reveal that the synthesis creates pyrrolic Fe(II)-plN3 as the active center whose flexibility manifested by being pulled out of the plane, enabled by defects, is crucial for collocating the reagents and allowing the chemistry to proceed. The catalyst catalyzes chemoselectively several substrates and possesses a unique trait whereby the chemistry is hindered for more acidic substrates than the hydrogen donors. This work paves the way toward noble-metal free single-atom catalysts for important chemical reactions.

A Slc38a8 Mouse Model of FHONDA Syndrome Faithfully Recapitulates the Visual Deficits of Albinism Without Pigmentation Defects.

We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.

Biosynthesis of Phomactin Platelet Activating Factor AntagonistRequires a Two-Enzyme Cascade.

Phomactin diterpenoids possess a unique bicyclo[9.3.1]-pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from amarine fungus,Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate-analogue FGGPP and elucidated the novel metal ion binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This work provides the first insights into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Biosynthesis of Phomactin Platelet Activating Factor Antagonist Requires a Two‐Enzyme Cascade

AbstractPhomactin diterpenoids possess a unique bicyclo[9.3.1]pentadecane skeleton with multiple oxidative modifications, and are good platelet‐activating factor (PAF) antagonists that can inhibit PAF‐induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from a marine fungus, Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate analogue FGGPP and elucidated the novel metal‐ion‐binding mode and enzymatic mechanism of PhmA through site‐directed mutagenesis. This study provides the first insight into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Uncovering the Dinuclear Mechanism of NO2-Involved NH3-SCR over Supported V2O5/TiO2 Catalysts.

Commercial vanadium oxide catalysts exhibit high efficiency for the selective catalytic reduction (SCR) of NO with NH3, especially in the presence of NO2 (i.e., occurrence of fast NH3-SCR). The high-activity sites and their working principle for the fast NH3-SCR reaction, however, remain elusive. Here, by combining in situ spectroscopy, isotopic labeling experiments, and density functional theory (DFT) calculations, we demonstrate that polymeric vanadyl species act as the main active sites in the fast SCR reaction because the coupling effect of the polymeric structure alters the elementary reaction step and effectively avoids the high energy barrier of the rate-determining step over monomeric vanadyl species. This study unveils the high-activity dinuclear mechanism of the NO2-involved SCR reaction over vanadia-based catalysts and provides a fundamental basis for developing high-efficiency and low V2O5-loading SCR catalysts.

Mechanism for Generating H2 O2 at Water-Solid Interface by Contact-Electrification.

The recent intensification of the study of contact-electrification at water-solid interfaces and its role in physicochemical processes lead to the realization that electron transfers during water-solid contact-electrification can drive chemical reactions. This mechanism, named contact-electro-catalysis (CEC), allows chemically inert fluorinated polymers to act like single electrode electrochemical systems. This study shows hydrogen peroxide (H2 O2 ) is generated from air and deionized water, by ultrasound driven CEC, using fluorinated ethylene propylene (FEP) as the catalyst. For a mass ratio of catalyst to solution of 1:10000, at 20°C, the kinetic rate of H2 O2 evolution reaches 58.87mmolL-1 gcat -1 h-1 . Electron paramagnetic resonance (EPR) shows electrons are emitted in the solution by the charged FEP, during ultrasonication. EPR and isotope labelling experiments show H2 O2 is formed from hydroxyl radicals (HO• ) or two superoxide radicals (O2 •- ) generated by CEC. Finally, it is traditionally believed such radicals migrate in the solution by Brownian diffusion prior to reactions. However, ab-initio molecular dynamic calculations reveal the radicals can react by exchanging protons and electrons through the hydrogen bonds network of water, i.e., owing to the Grotthuss mechanism. This mechanism can be relevant to other systems, artificial or natural, generating H2 O2 from air and water.

Microbial degradation and assimilation of veratric acid in oxic and anoxic groundwaters.

Microbial communities are key players in groundwater ecosystems. In this dark environment, heterotrophic microbes rely on biomass produced by the activity of lithoautotrophs or on the degradation of organic matter seeping from the surface. Most studies on bacterial diversity in groundwater habitats are based on 16S gene sequencing and full genome reconstructions showing potential metabolic pathways used in these habitats. However, molecular-based studies do not allow for the assessment of population dynamics over time or the assimilation of specific compounds and their biochemical transformation by microbial communities. Therefore, in this study, we combined DNA-, phospholipid fatty acid-, and metabolomic-stable isotope probing to target and identify heterotrophic bacteria in the groundwater setting of the Hainich Critical Zone Exploratory (CZE), focusing on 2 aquifers with different physico-chemical conditions (oxic and anoxic). We incubated groundwater from 4 different wells using either 13C-labeled veratric acid (a lignin-derived compound) (single labeling) or a combination of 13CO2 and D-labeled veratric acid (dual labeling). Our results show that heterotrophic activities dominate all groundwater sites. We identified bacteria with the potential to break down veratric acid (Sphingobium or Microbacterium). We observed differences in heterotrophic activities between the oxic and anoxic aquifers, indicating local adaptations of bacterial populations. The dual labeling experiments suggested that the serine pathway is an important carbon assimilation pathway and that organic matter was an important source of hydrogen in the newly produced lipids. These experiments also yielded different labeled taxa compared to the single labeling experiments, showing that there exists a complex interaction network in the groundwater habitats.

Mechanistic Characterisation of Collinodiene Synthase, a Diterpene Synthase from Streptomyces collinus.

Two homologs of the diterpene synthase CotB2 from Streptomyces collinus (ScCotB2) and Streptomyces iakyrus (SiCotB2) were investigated for their products by in vitro incubations of the recombinant enzymes with geranylgeranyl pyrophosphate, followed by compound isolation and structure elucidation by NMR. ScCotB2 produced the new compound collinodiene, besides the canonical CotB2 product cyclooctat-9-en-7-ol, dolabella-3,7,18-triene and dolabella-3,7,12-triene, while SiCotB2 gave mainly cyclooctat-9-en-7-ol and only traces of dolabella-3,7,18-triene. The cyclisation mechanism towards the ScCotB2 products and their absolute configurations were investigated through isotopic labelling experiments.