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Access to the oxygen-evolving complex in photosynthesis is restricted by specific barriers in the channels connecting the Mn 4 CaO 5 catalyst with bulk water. Together with other recent data, this finding allows assigning the two substrate waters.
The present work quantifies metal-contaminant interactions between palladium substrates and three salient chlorinated organic contaminants, namely trichloroethylene 1,3,5-trichlorobenzene (TCB), and 3,3′,4,4′-tetrachlorobiphenyl (PCB77). Given that Pd is one of the conventional catalytically active materials known for contaminant removal, maximizing catalytic efficiency through optimal adsorption dynamics reduces the cost of remediation of contaminants that are persistent water pollutants chronically affecting public health. Adsorption efficiency analyses from all-atom molecular dynamics (MD) simulations advance the understanding of reaction mechanisms available from density functional theory (DFT) calculations to an extractable feature scale that can fit the parametric design of supported metal catalytic systems and feed into high throughput catalyst selection. Data on residence time, site-specific adsorption, binding energies, packing geometries, orientation profiles, and the effect of adsorbate size show the anomalous behaviour of organic contaminant adsorption on the undercoordinated {110} surface as compared to the {111} and {100} surfaces. The intermolecular interaction within contaminants from molecular dynamics simulation exhibits refreshing results than ordinary single molecule density functional theory calculation. Since complete adsorption and dechlorination is an essential step for chlorinated organic contaminant remediation pathways, the presented profiles provide essential information for designing efficient remediation systems through facet-controlled palladium nanoparticles.
Solutions to the inverse heat conduction problem (IHCP) are methods that can be used to quantify surface heat flux in multi-layer materials for components in which there are limited subsurface (internal) temperature measurements, such as coated components. A critical consideration is to capture high frequency fluctuations using a practical heat flux sensor. To that end, this paper highlights key parameters for calculating accurate surface heat transfer. Specifically, this research extends the available solutions to the IHCP for multi-substrate structures through an impulse response methodology. The sensitivity of the impulse method was quantified with respect to practical measurements. Further, when compared to the inverse case, the impulse method resulted in lower errors when calculating surface heat flux over a range of conditions. Overall, this work provides a foundation for deducing heat flux from a subsurface heat flux sensor while maintaining a high-frequency response.
Oxidative modifications of protein cysteines play a crucial role in regulating redox homeostasis, redox signaling, and protein function. One of the modifications involves the oxidation of a cysteine thiol to a sulfinic acid (R-SO 2 H) which has been considered a marker for oxidative stress and an important activator for certain proteins. Here, in this study, we report a protocol for the selective formation of sulfinic acids on cysteine residues in a simple 3-step operation under mild conditions: thiol-blocking, oxidation, and reduction. The specificity and efficiency of the method were demonstrated by using both small molecule substrates and a protein model.
The genomes of anaerobic gut fungi (AGF) encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been experimentally validated or expressed in heterologous systems. Here, we developed a predictive bioinformatic pipeline to annotate novel putative CAZymes from anaerobic fungi and validate their activity through large-scale heterologous expression in Escherichia coli. A total of 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of the total proteome using high-throughput proteomic screening. Among these 17 heterologously expressed proteins, analysis with AlphaFold and FoldSeek predicted 13 multi-functional proteins containing catalytic domains fused with repetitive fungal dockerins, and half of the substrate predictions were experimentally validated. One promising enzyme, celsome_012, exhibited robust and specific activity against beechwood xylan at 37°C and pH 6.4, with titers that were also fivefold higher than those of other recombinant proteins screened here. Both Michaelis-Menten kinetics and the linearized Lineweaver-Burk equation yielded consistent values for K m , and its activation energy was estimated at 51.9 kJ/mol based on the Arrhenius model. This work supports the industrial translation of anaerobic fungal CAZymes due to their robust lignocellulolytic activity and provides a framework for prioritizing AGF proteins for efficient E. coli heterologous expression.
The goal of this project is to build a multimodal optical microscope to measure the binding, processive degradation, and pausing behaviors of cellulases as they interact with and degrade both synthetic and naturally occurring lignocellulosic walls. To achieve this, we will use high spatio-temporal single-molecule imaging to track cellulases, while visualizing specific molecular components of cellulose, lignin and hemicellulose, that make up their lignocellulose substrate. The microscope will combine Interferometric Scattering (iSCAT), which provides unprecedented spatiotemporal resolution; Total Internal Reflection Fluorescence (TIRF), which provides single-molecule resolution of multiple fluorophore-labeled molecules; and Stochastic Reconstruction (STORM), which allows for three-dimensional super-resolution imaging of intact plant cell walls during degradation. Initial studies will investigate cellulase dynamics on in vitro-assembled cell wall analogs, and later work will progress to using native plant cell walls.
Polypeptide scaffolds comprising enzymatic proteins are provided. The enzymatic polypeptide scaffolds comprise heterologous enzymes to form a heterologous metabolic pathway, and can be targeted to a substrate through a surface anchoring domain. The enzymatic polypeptide scaffolds leverage the high specificity and affinity protein/protein interaction between the cohesins and dockerins of microorganismal cellulosomes to form custom enzymatic arrays.
Additive manufacturing (AM) of high-strength aluminum alloys can produce high-performance aerospace and biomedical parts with unique geometries and functions. However, many high-performance alloy systems such as AA 7075 pose challenges during AM due to thermal cycles that cause hot cracking, limiting AM to casting-friendly alloys. Stock AA 7075 alloy poses additional challenges due to the excessive growth of insoluble precipitates during casting and needs to be thermomechanically processed to obtain acceptable mechanical properties. Here, this work uses liquid metal jet printing to produce high-resolution pure AA 7075 parts at high strength (593 MPa) and ductility (10 pct), surpassing the specific strength of any type of steel. The parts are printed by depositing fine droplets on a high-temperature substrate that reduces cooling rates, preventing hot cracking. The results show that fully dense, crack-free parts can only be printed at substrate temperatures of 500 °C with a cooling rate threshold of < 2000 K/s to prevent cracking and < 500 K/s to eliminate porosity. The tensile strength, microhardness, and ductility of the parts matched or were beyond the wrought alloy specifications after heat treatment, possibly due to high solidification rates that produce finer insoluble precipitates. Our results indicate that many challenging high-performance alloy systems can now be 3D printed based on the critical thresholds for cooling rates using any AM method that can satisfy these conditions.
Thermite reactions –highly energetic redox processes between a metal and an oxide—are used in welding, propulsion, and the fabrication of advanced materials. When reduced to the nanoscale, these reactions exhibit enhanced energetic performance, but their ultrafast dynamics remain poorly understood. Gaining insight into charge transfer during these processes is essential for advancing applications in energy conversion and materials design. Here we show that the reaction between aluminium and hematite, a common iron oxide, can be tracked with femtosecond resolution using extreme ultraviolet (EUV) time-resolved absorption spectroscopy at the Fe M 2,3 and Al L 2,3 edges. By exciting the system with an ultrashort optical pulse and probing element-specific absorption changes, we observe an early spectral shift that reveals the formation of localized charge carriers (polarons). Comparing samples with different supporting substrates highlights ultrafast electron transfer from aluminium to hematite. These results demonstrate an approach to investigating charge flow in energetic materials and provide a basis for studying fast chemical reactions with chemical specificity.
2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO 2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H + and CO 2 to favor the metabolically preferred carboxylation product, acetoacetate.
Proton-coupled electron transfer (PCET) reagents have emerged as powerful tools for transferring net H atoms to organic substrates from relatively weak X–H bonds. One advantage of employing PCET reagents is the tunability of the X–H bond strength by independently varying their redox potential and/or p K a for selective substrate reductions; however, the rational development of modular catalytic PCET reagents based on these features remains underdeveloped. In this work, we address important mechanistic questions relevant to a dimethylaniline-appended cobaltocene PCET mediator that our lab has previously introduced. Specifically, we examine where protonation occurs within the reactive Co(II, NH) + intermediate of a Brønsted-base modified cobaltocene mediator, whether substrate reduction and hydrogen evolution reaction (HER) proceed by a common or bifurcated mechanistic pathway, and how the redox, acid–base, and structural properties of PCET mediators can dictate their reactivity and selectivity. We show that substrate compatibility can be tuned and, via a model study with N -aryl imine substrates, provide data pointing to a multisite PCET (MS-PCET) pathway. Moreover, we rigorously characterize the site of protonation in the reactive reduced, protonated form of the mediator, and through kinetic analysis establish that the pathway for undesired competing HER is fundamentally different and involves Cp-ring protonation. Our findings point to a high degree of flexibility in the design of reductive PCET mediators.
Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.
ABSTRACT We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta -cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase’s ability to cleave both 2-aminophenol and catechol in vitro , in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta -cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.
Soil dwelling microorganisms are key players in the terrestrial carbon cycle, driving both the degradation and stabilization of soil organic matter. Bacterial community structure and function vary with respect to land use; yet the ecological drivers of this variation remain poorly described and difficult to predict. For this work, we conducted a multi-substrate DNA-stable isotope probing experiment across cropland, old-field, and forest habitats to link carbon mineralization dynamics with the dynamics of bacterial growth and carbon assimilation. We tracked the movement of 13 C derived from five distinct carbon sources as it was assimilated into bacterial DNA over time. We show that carbon mineralization, community composition, and carbon assimilation dynamics all differed with respect to land use. We also show that microbial community dynamics affect carbon assimilation dynamics and are associated with soil DNA content. Soil DNA yield is easy to measure and may be useful in predicting microbial community dynamics linked to soil carbon cycling. Soil dwelling microorganisms are key players in the terrestrial carbon cycle, driving both the degradation and stabilization of soil organic matter. Microbial communities vary with respect to land use, but we still have an incomplete understanding of how variation in community structure links to variation in community function. DNA stable isotope probing (DNA-SIP) is a high-resolution method that can identify specific microbial taxa that assimilate carbon in situ. We conducted a large-scale multi-substrate DNA-SIP experiment to explore differences in bacterial activity across land-use regimes. We show that microbial community dynamics vary with land use, that these dynamics are linked to soil carbon cycling, and that they are associated with easily measured soil properties.
Isoprenoid compounds are biologically ubiquitous, and their characteristic modularity has afforded products ranging from pharmaceuticals to biofuels. Isoprenoid production has been largely successful in Escherichia coli and Saccharomyces cerevisiae with metabolic engineering of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways coupled with the expression of heterologous terpene synthases. Yet conventional microbial chassis pose several major obstacles to successful commercialization including the affordability of sugar substrates at scale, precursor flux limitations, and intermediate feedback-inhibition. Now, recent studies have challenged typical isoprenoid paradigms by expanding the boundaries of terpene biosynthesis and using non-model organisms including those capable of metabolizing atypical C1 substrates. Conversely, investigations of non-model organisms have historically informed optimization in conventional microbes by tuning heterologous gene expression. Here, we review advances in isoprenoid biosynthesis with specific focus on the synergy between model and non-model organisms that may elevate the commercial viability of isoprenoid platforms by addressing the dichotomy between high titer production and inexpensive substrates.
Abstract cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.
Yttrium iron garnet (Y 3 Fe 5 O 12 ; YIG) has a unique combination of low magnetic damping, high spin-wave conductivity, and insulating properties that make it a highly attractive material for a variety of applications in the fields of magnetics and spintronics. While the room-temperature magnetization dynamics of YIG have been extensively studied, there are limited reports correlating the low-temperature magnetization dynamics to the material structure or growth method. Here, in this study, we investigate liquid phase epitaxy grown YIG films and their magnetization dynamics at temperatures down to 10 K. We show there is a negligible increase in the ferromagnetic resonance linewidth down to 10 K, which is unique when compared with YIG films grown by other deposition methods. From the broadband ferromagnetic resonance measurements, polarized neutron reflectivity, and scanning transmission electron microscopy, we conclude that these liquid phase epitaxy grown films have negligible rare-earth impurities present, specifically the suppression of Gd diffusion from the Gd 3 Ga 5 O 12 (GGG) substrate into the Y 3 Fe 5 O 12 film, and therefore negligible magnetic losses attributed to the slow-relaxation mechanism. Overall, liquid phase epitaxy YIG films have a YIG/GGG interface that is five times sharper and have ten times lower ferromagnetic resonance linewidths below 50 K than comparable YIG films by other deposition methods. Thus, liquid phase epitaxy grown YIG films are ideal for low-temperature experiments/applications that require low magnetic losses, such as quantum transduction and manipulation via magnon coupling.
This data package is associated with the publication “Modeling Microbial Regulatory Feedback in Organic Matter Decomposition Identifies Copiotrophic Traits as Key Drivers of Positive Priming” published as a preprint on BioRXiv by Ahamed et al. (2026); https://doi.org/10.1101/2024.08.11.607483. The package contains MATLAB scripts and saved simulation outputs used to implement a cybernetic model of microbial regulation during complex organic matter (OM) decomposition governing priming effects. It includes models of (i) single microbial functional groups (copiotrophic or oligotrophic degraders) and (ii) binary consortia composed of degraders and non-degraders with contrasting or common growth traits. Simulation results were generated using Monte Carlo analyses, with randomized key model parameters across a range of environmental mixing fractions of complex and labile OM. The dataset was created to provide a transparent and reusable computational framework for systematically exploring how microbial growth traits, metabolic regulation, and community composition influence OM decomposition dynamics and priming effects. For details on how to navigate data packages generated by this project, see https://data.ess-dive.lbl.gov/portals/PNNLRiverCorridorSFA/About. In addition to a readme, this data package also includes a file-level metadata (FLMD) file that describes each file and a data dictionary (DD) that describes the variable definitions. This package includes: (1) annotated MATLAB code implementing the system of ordinary differential equations and cybernetic control laws; (2) saved output files containing data (e.g., biomass, substrates, enzyme levels, priming metrics); and (3) scripts for processing saved outputs and regenerating figures. Specifically, the data package contains three main MATLAB scripts: runPrimingModel.m, runPlotData.m, and runPlotSuppFigS1.m, along with this readme and supporting documentation. Users should begin with runPrimingModel.m, which contains the annotated code implementing the system of ordinary differential equations and cybernetic control laws. This script runs the Monte Carlo simulations of microbial OM decomposition and allows users to modify microbial trait definitions, adjust parameter distributions, or define new community configurations. Simulation outputs are automatically saved as .mat files in the folder named SavedData, which stores all pre-generated results included in this package. The second script, runPlotData.m, reads files from the SavedData folder and processes them to regenerate the figures presented in the manuscript. The third script, runPlotSuppFigS1.m, specifically generates Figure S1 in the Supplementary Material of the manuscript. The package also includes the aforementioned files in non-proprietary .txt format. If users intend to use them, they should first save the files in their respective .m or .mat formats prior to execution in MATLAB.