Enzymatic Reaction Research Articles

Calibration-Free and Highly Sensitive Potentiometric Enzyme-Based Biosensors.

Enzyme-based amperometric biosensors have become popular for healthcare applications. However, they have been under constant pressure for technological innovation to improve their sensitivity and usability. An ideal biosensor has high sensitivity and calibration-free characteristics. This study aims to report enzyme-based glucose and lactate sensors that utilize a proposed "time-derivative of potential (dOCP/dt)" method, with a further aim being to prove theoretically and experimentally that dOCP/dt values are proportional to substrate concentration. High sensitivity is obtained regardless of the electrode size because the electrode potential is independent of the electrode area in the biosensor. Importantly, because the substrate diffusion determines the enzyme reaction rate on the sensors, the dOCP/dt biosensors can essentially eliminate external influences such as temperature and pH. The result is the successful realization of a biosensor that is calibration-free, making it a much more practical option.

Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-β-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min−1 mg−1.

Reshaping and enzymatic activity may allow viruses to move through the mucus.

Filamentous viruses like influenza and torovirus often display systematic bends and arcs of mysterious physical origin. We propose that such viruses undergo an instability from a cylindrically symmetric to a toroidally curved state. This "toro-elastic" state emerges via spontaneous symmetry breaking under prestress due to short range spike protein interactions magnified by surface topography. Once surface stresses are sufficiently large, the filament buckles and the curved state constitutes a soft mode that can potentially propagate through the filament's material frame around a Mexican-hat-type potential. In the mucus of our airways, which constitutes a soft, porous 3D network, glycan chains are omnipresent and influenza's spike proteins are known to efficiently bind and cut them. We next show that such a non-equilibrium enzymatic reaction can induce spontaneous rotation of the curved state, leading to a whole body reshaping propulsion similar to - but different from - eukaryotic flagella and spirochetes.

Substituting partial chemical nitrogen fertilizers with organic fertilizers maintains grain yield and increases nitrogen use efficiency in maize

IntroductionLong-term application of excessive nitrogen (N) not only leads to low N use efficiency (NUE) but also exacerbates the risk of environmental pollution due to N losses. Substituting partial chemical N with organic fertilizer (SP) is an environmentally friendly and sustainable fertilization practice. However, the appropriate rate of SP in rainfed maize cropping systems in semi-arid regions of China is unknown.MethodsTherefore, we conducted a field experiment between 2021 and 2022 in a semi-arid region of Northern China to investigate the effects of SP on maize growth, carbon and N metabolism (C/NM), and NUE. The following treatments were used in the experiment: no N application (CK), 100% chemical N (SP0, 210 kg N ha–1), and SP substituting 15% (SP1), 30% (SP2), 45% (SP3), and 60% (SP4) of the chemical N. The relationship between these indicators and grain yield (GY) was explored using the Mantel test and structural equation modeling (SEM).Results and discussionThe results found that the SP1 and SP2 treatments improved the assimilates production capacity of the canopy by increasing the leaf area index, total chlorophyll content, and net photosynthetic rate, improving dry matter accumulation (DMA) by 6.2%–10.6%, compared to the SP0 treatment. SP1 and SP2 treatments increased total soluble sugars, starch, free amino acids, and soluble protein contents in ear leaves via increasing the enzymatic reactions related to C/NM in ear leaves during the reproductive growth stage compared with SP0 treatment. The highest plant nitrogen uptake (PNU) and nitrogen recovery efficiency were obtained under the SP2 treatment, and the GY and nitrogen agronomic efficiency were higher than the SP0 treatment by 9.2% and 27.8%. However, SP3 and SP4 treatments reduced DMA and GY by inhibiting C/NM in ear leaves compared to SP0 treatment. Mantel test and SEM results revealed that SP treatments indirectly increased GY and PNU by directly positively regulating C/NM in maize ear leaves. Therefore, in the semi-arid regions, substituting 30% of the chemical N with SP could be considered. This fertilizer regime may avoid GY reduction and improve NUE. This study provides new insights into sustainable cultivation pathways for maize in semi-arid regions.

Abstract C050: Vitamin B6 restriction impairs pancreatic cancer progression

Abstract Introduction: Cancer cells utilize altered metabolism to sustain their rapid growth, and understanding how these metabolic changes aid in tumor development could enhance current treatment approaches. Vitamins are essential micronutrients necessary in small quantities to support the proper functioning and metabolism of our organs and systems. Despite the recognized importance of vitamins in maintaining overall health, there is no direct evidence linking vitamin metabolism to pancreatic ductal adenocarcinoma (PDAC). Methods: PDAC animal models, organoid models, 3D-culture models, and LC-MS/MS-based metabolomic analysis were used to explore the role of vitamin B6 (VB6) in pancreatic cancer. Results: Our data showed that 1) Patients with PDAC exhibit lower levels of circulating vitamin B6 compared to healthy individuals of the same age. Additionally, vitamin B6 levels are reduced in the interstitial fluid of KPC (KrasG12D/+; p53R172H/+; Pdx1-Cretg/+) tumors and in the blood of mice with KPC tumors compared to healthy mice of the same age; 2) PDAC cells metabolize VB6 more rapidly than non-cancerous pancreatic epithelial cells; 3) VB6 restriction impairs PDAC cell proliferation in vitro and PDAC tumor growth in vivo; 4) VB6 depletion decreases key metabolites of the one-carbon pathway necessary for PDAC cell growth; 5) Inhibiting the one- carbon pathway mimics the effects of a vitamin B6 deficiency on PDAC cell proliferation. And knocking down VB6-dependent enzymes SHMT1/2 negates the pro-growth impact of VB6. Conclusion: Our research suggests that VB6 plays a key role in regulating various metabolic processes essential for the growth of PDAC cells. By focusing on inhibiting VB6-dependent enzymatic reactions, we may uncover new therapeutic strategies for treating PDAC. Citation Format: Chunbo He, Kamiya Mehla. Vitamin B6 restriction impairs pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C050.

Microbial synthesis of sedoheptulose from glucose by metabolically engineered Corynebacterium glutamicum

BackgroundSeven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production.ResultsIn this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production.ConclusionWe achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.

192 Prediction of fecal starch content of fattening cattle using near-infrared spectroscopy and machine learning

Abstract Starch content in cattle feces can be used to predict starch digestibility. Near-infrared spectroscopy (NIRS) can be used to rapidly and non-chemically predict fecal starch content after a calibration model has been consequentially developed. Recent advancements in machine learning have introduced potent methods for developing optimized models. In addition, diverse preprocessing methods, such as scatter correction and smoothing, are available to eliminate irrelevant variance sources. This study aimed to identify optimal spectral preprocessing and regression methods for predicting fecal starch content using NIR spectra. NIR spectra were measured for 196 fecal samples collected from 9 fattening cattle farms. Fecal starch content on a DM basis was measured using enzymatic reactions and colorimetry. The 196 samples were divided into a calibration set (n = 123), a validation set (n = 29) comprising four farms, and an external validation set (n = 44) comprising five farms that differed from the calibration set. Based on the calibration set, two preprocessing methods were selected using the partial least squares (PLS) model. In method 1, the calibration models were developed by a grid search 5-fold cross validation on the 1,152 preprocessed calibration sets, and preprocessing with the lowest root mean square error (RMSE) of cross-validation among these calibration sets was selected. In method 2, the original calibration set (n = 123) was divided into a calibration set (n = 116) and a preprocessing selection set (n = 7) with different neutral detergent fiber and similar starch contents. After preprocessing, calibration models were developed as described in method 1. The preprocessing selection sets were then predicted by the developed models, and preprocessing with the lowest RMSE among these preprocessing selection sets was selected. Using the preprocessing methods selected in methods 1 and 2, the validation and external validation sets were predicted using seven regression models. As a result, the preprocessing selected by method 1 was in the wavelength range of 400–2500 nm, with robust normal variate (RNV) scatter correction and first-order derivative Savitzky-Golay filtering (2nd order polynomial, 2-nm window size). The preprocessing selected by method 2 had a wavelength range of 1500–2500 nm, RNV scatter correction, and 2nd-order derivative Savitzky-Golay filtering (3rd order polynomial, 25-nm window size). Among the seven regression models, the Lasso model was the most accurate for prediction. The Lasso models based on the preprocessing methods selected by methods 1 and 2 had RMSE of external validation = 2.054% of DM and 1.540% of DM, respectively. The feature importance of the Lasso model based on preprocessing method selected by methods 2 was higher at wavelengths of 1775 nm and 2325 nm. In conclusion, seven models were developed and evaluated based on diverse preprocessing methods using NIRS. The Lasso model exhibited the highest accuracy.

Efficient Encapsulation of β-Lapachone into Self-Immolative Polymer Nanoparticles for Cyclic Amplification of Intracellular Reactive Oxygen Species Stress.

The selective upregulation of intracellular oxidative stress in cancer cells presents a promising approach for effective cancer treatment. In this study, we report the integration of enzyme catalytic amplification and chemical amplification reactions in β-lapachone (Lap)-loaded micellar nanoparticles (NPs), which are self-assembled from reactive oxygen species (ROS)-responsive self-immolative polymers (SIPs). This integration enables cyclic amplification of intracellular oxidative stress in cancer cells. Specifically, we have developed ROS-responsive SIPs with phenylboronic ester triggering motifs and hexafluoroisopropanol moieties in the side chains, significantly enhancing Lap loading efficiency (98%) and loading capacity (33%) through multiple noncovalent interactions. Upon ROS activation in tumor cells, the Lap-loaded micellar NPs disassemble, releasing Lap and generating additional ROS via enzyme catalytic amplification. This process elevates intracellular oxidative stress and triggers polymer depolymerization in a positive feedback loop. Furthermore, the degradation of SIPs via chemical amplification produces azaquinone methide intermediates, which consume intracellular thiol-related substrates, disrupt intracellular redox hemostasis, further intensify oxidative stress, and promote cancer cell apoptosis. This work introduces a strategy to enhance intracellular oxidative stress by combining enzymatic and chemical amplification reactions, providing a potential pathway for the development of highly efficient anticancer agents.

A urease-based pH photoswitch: a general route to light-to-pH transduction.

New approaches for the integration of chemical and physical stimuli to control the dynamics of artificial enzymatic reaction networks (ERNs) are needed. Here, we present a general approach to convert a light stimulus into a time-programmed pH response. We developed and characterized a panel of photoswitchable inhibitors of urease. Urease activity, now regulated by light via the photoinhibitors, leads to an increase in pH upon hydrolysis of urea into ammonia. Careful choice of characteristics of light, and concentrations of enzyme, substrate, and photoinhibitor, allowed us to control the timing of the pH transition. Furthermore, as all enzymes have an activity-pH profile, the urease photoinhibitor system can be used to regulate the activities of other enzymes in small reaction networks.

Dual-pore protocells with multitasking capacities for simultaneous delivery of therapeutic enzymes and drugs in macrophage depletion therapy.

Macrophages are usually present in solid tumors where they participate in tumor progression, angiogenesis, immunosuppression and metastasis. The design of nanocarriers capable of delivering therapeutic agents to specific cell populations has received considerable attention in the last decades. However, the capacity of many of these nanosystems to deliver multiple therapeutic agents with very different chemical properties is more limited. Herein, a novel multitasking nanoplatform capable of delivering large macromolecules and cytotoxic drugs to macrophages is presented. This novel nanosystem has exhibited excellent skills in performing simultaneous tasks, macrophage depletion and glucose starvation, maintaining the oxygen levels in the tissue. This nanodevice is composed of a dual-pore mesoporous silica core with the capacity to house small cytotoxic drugs, such as doxorubicin or zoledronic acid, and large macromolecules, such as glucose oxidase. The external surface of the silica core was coated with a lipid bilayer to avoid the premature release of the housed drugs. Finally, polymeric nanocapsules loaded with catalase were covalently anchored on the outer lipid bilayer, and carboxy-mannose was attached to the exposed side of the nanocapsules to provide selectivity to the macrophages. These nanoassemblies were able to transport enzymes (Gox and CAT), maintaining their catalytic activity. Therefore, they could induce glucose starvation, keeping the oxygen levels in the tissue, owing to the tandem enzymatic reaction. The capacity of these nanoassemblies to deliver therapeutic agents to macrophages was evaluated both in static and under flow conditions, showing a rapid capture of the nanoparticles by the macrophages. Once there, the nanoassemblies also exhibited excellent capacity to induce potent macrophage depletion. This strategy can be directly adapted for the treatment of different malignancies due to the modular nature of the nanoplatform, which can be loaded with different therapeutic agent combinations and pave the way for the development of personalized nanomedicines for diverse types of tumors.

In enzymatic reactions, the reverse reaction reduces product noise

Enzymatic reactions are essential for most cellular reactions and ubiquitous in living organisms. In the present study, we explore the pivotal role of the reverse reaction in enzymatic reactions. It is a powerful noise-buffering motif. By SSA (stochastic simulation algorithm), a remarkable 32% reduction of product CV (coefficient of variation) was observed. To better understand the causes, we split the upstream noise. The product CV reduction is more than 35% for the noise inherited from the enzyme but merely 6%–21% for that from the substrate. It implies that the system applies different strategies to different upstream noises. We identified two leading causes responsible for noise attenuation. A cell is well designed to control its intracellular noise, and to acquire wisdom from nature is always enjoyable.

High performance computational approach to study model describing reversible two-step enzymatic reaction with time fractional derivative

Enzyme reactions have numerous applications in diverse disciplines of science like chemistry, biology and biomechanics. In this study, we examine the role and act of enzymes in chemical reactions which is considered in the frame of fractional order model. The proposed model includes system of four equations which are studied via Caputo fractional operator. The systems of non-linear equations are evaluated by a semi-analytical approach called q\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$q$$\\end{document}-homotopy analysis transform method. The uniqueness and existence of the solutions has been investigated through fixed point theorem. The solutions of the proposed model are achieved through the considered method and the obtained outcomes are in the form of series which shows rapid convergence. The solutions are computed and graphs are plotted for the obtained results using mathematica software. The achieved results by the proposed method are unique and illustrate the significant dynamics of the considered model via 3D plots and graphs. The results of this study demonstrate the importance and effectiveness of projected derivative and technique in the analysis of time dependent fractional mathematical models. This study also gives an idea to extend the applications of enzymatic reactions in drug development, bio mechanics, and chemical reactions in various cellular metabolisms. Also, enzymatic reactions have a vital role in the fields of the food industry for processing food, in biotechnology for the manufacture of biofuels, and in metabolic engineering to design metabolic pathways.

D-Band Center Engineered Pt-Based Nanozyme for Personalized Pharmacovigilance.

A high-efficiency PtZnCd nanozyme was screened with density functional theory (DFT) and unique d-orbital coupling features for sensitive enrichment and real-time analysis of CO-releasing molecule-3 (CORM-3). Multi-catalytic sites in the nanozyme showed a high reactivity of up to 72.89 min-1 for peroxidase-like enzymes (POD) reaction, which was 2.2, 4.07, and 14.67 times higher than that of PtZn (32.67 min-1), PtCd (17.89 min-1), and Pt (4.97 min-1), respectively. Normalization of the catalytic sites showed that the catalytic capacity of the active site in PtZnCd was 2.962 U μmol-1, which was four times higher than that of pure Pt site (0.733 U μmol-1). DFT calculations showed that improved d-orbital coupling between different metals reduces the position of the center of the shifted whole d-band relative to the Fermi energy level, thereby increasing the contribution of the sites to the electron transfer from the active center, accompanied with enhanced substrate adsorption and intermediate conversion in the catalytic process. The potential adsorption principle and color development mechanism of CORM-3 on PtZnCd were determined, and the practical application in drug metabolism was validated in vitro, in zebrafish and mice as a model, demonstrating that transition metal doping effectively engineers high-performance nanozymes and optimizes artificial enzymes.

D‐Band Center Engineered Pt‐Based Nanozyme for Personalized Pharmacovigilance

A high‐efficiency PtZnCd nanozyme was screened with density functional theory (DFT) and unique d‐orbital coupling features for sensitive enrichment and real‐time analysis of CO‐releasing molecule‐3 (CORM‐3). Multi‐catalytic sites in the nanozyme showed a high reactivity of up to 72.89 min‐1 for peroxidase‐like enzymes (POD) reaction, which was 2.2, 4.07, and 14.67 times higher than that of PtZn (32.67 min‐1), PtCd (17.89 min‐1), and Pt (4.97 min‐1), respectively. Normalization of the catalytic sites showed that the catalytic capacity of the active site in PtZnCd was 2.962 U μmol‐1, which was four times higher than that of pure Pt site (0.733 U μmol‐1). DFT calculations showed that improved d‐orbital coupling between different metals reduces the position of the center of the shifted whole d‐band relative to the Fermi energy level, thereby increasing the contribution of the sites to the electron transfer from the active center, accompanied with enhanced substrate adsorption and intermediate conversion in the catalytic process. The potential adsorption principle and color development mechanism of CORM‐3 on PtZnCd were determined, and the practical application in drug metabolism was validated in vitro, in zebrafish and mice as a model, demonstrating that transition metal doping effectively engineers high‐performance nanozymes and optimizes artificial enzymes.

Proteogenomic reconstruction of organ-specific metabolic networks in an environmental sentinel species, the amphipod Gammarus fossarum

Metabolic pathways are affected by the impacts of environmental contaminants underlying a large variability of toxic effects across different species. However, the systematic reconstruction of metabolic pathways remains limited in environmental sentinel species due to the lack of available genomic data in many taxa of animal diversity. In this study we used a multi-omics approach to reconstruct the most comprehensive map of metabolic pathways for a crustacean model in biomonitoring, the amphipod Gammarus fossarum in order to improve the knowledge of the metabolism of this sentinel species.We revisited the assembly of RNA-seq data by de novo approaches to reduce RNA contaminants and transcript redundancy. We also acquired extensive mass spectrometry shotgun proteomic data on several organs from a reference population of G. fossarum males and females to identify organ-specific metabolic profiles.The G. fossarum metabolic pathway reconstruction (available through the metabolic database GamfoCyc) was performed by adapting the genomic tool CycADS and we identified 377 pathways representing 7630 annotated enzymes, 2610 enzymatic reactions and the expression of 858 enzymes was experimentally validated by proteomics. To our knowledge, our analysis provides for the first time a systematic metabolic pathway reconstruction and the proteome profiles of these pathways at the organ level in this sentinel species. As an example, we show an elevated abundance in enzymes involved in ATP biosynthesis and fatty acid beta-oxidation indicative of the high-energy requirement of the gills, or the key anabolic and detoxification role of the hepatopancreatic caeca, as exemplified by the specific expression of the retinoid biosynthetic pathways and glutathione synthesis.In conclusion, the multi-omics data integration performed in this study provides new resources to investigate metabolic processes in crustacean amphipods and their role in mediating the effects of environmental contaminant exposures in sentinel species. SynopsisThis study provide the first evidence that it is possible to combine multiple omics data to exhaustively describe the metabolic network of a model species in ecotoxicology, Gammarus fossarum, for which a reference genome is not yet available.

The aroma transformation of Japanese sea bass (Lateolabrax japonicas) through endogenous enzyme incubation during the lag phase of attached microorganisms

Endogenous enzymes play a crucial role in determining fish product aroma. However, the attached microorganisms can promote enzyme production, making it challenging to identify specific aromatic compounds resulting from endogenous enzymes. Thus, we investigated the aroma transformation of Japanese sea bass through enzymatic incubation by controlling attached microorganisms during the lag phase. Our results demonstrate that enzymatic incubation significantly enhances grassy and sweet notes while reducing fishy odors. These changes in aroma are associated with increased levels of 10 volatile compounds and decreased levels of 3 volatile compounds. Among them, previous studies have reported enzyme reaction pathways for octanal, 1-nonanal, vanillin, indole, linalool, geraniol, citral, and 6-methyl-5-hepten-2-one; however, the enzymatic reaction pathways for germacrene D, beta-caryophyllene, pristane, 1-tetradecene and trans-beta-ocimene remain unclear. These findings provide novel insights for further study to elucidate the impact of endogenous enzymes on fish product aromas.

BayesianSSA: a Bayesian statistical model based on structural sensitivity analysis for predicting responses to enzyme perturbations in metabolic networks

BackgroundChemical bioproduction has attracted attention as a key technology in a decarbonized society. In computational design for chemical bioproduction, it is necessary to predict changes in metabolic fluxes when up-/down-regulating enzymatic reactions, that is, responses of the system to enzyme perturbations. Structural sensitivity analysis (SSA) was previously developed as a method to predict qualitative responses to enzyme perturbations on the basis of the structural information of the reaction network. However, the network structural information can sometimes be insufficient to predict qualitative responses unambiguously, which is a practical issue in bioproduction applications. To address this, in this study, we propose BayesianSSA, a Bayesian statistical model based on SSA. BayesianSSA extracts environmental information from perturbation datasets collected in environments of interest and integrates it into SSA predictions.ResultsWe applied BayesianSSA to synthetic and real datasets of the central metabolic pathway of Escherichia coli. Our result demonstrates that BayesianSSA can successfully integrate environmental information extracted from perturbation data into SSA predictions. In addition, the posterior distribution estimated by BayesianSSA can be associated with the known pathway reported to enhance succinate export flux in previous studies.ConclusionsWe believe that BayesianSSA will accelerate the chemical bioproduction process and contribute to advancements in the field.

Theoretical Framework for Novel Catalytic Biomolecules Composed of Multiple Peptides.

Protein-based enzymes are among the most efficient catalysts on our planet. A common feature of protein enzymes is that all catalytic amino acids occupy a limited, narrow space and face each other. In this study, we created a theoretical novel biomimetic molecule containing different multiple catalytic peptides. Although single peptides are far less catalytically efficient than protein enzymes, Octopus-arms-mimicking biomolecules containing eight different peptides (Octopuzymes) can efficiently catalyze organic reactions. Since structural information for extant protein enzymes, predicted enzymes based on genome data, and artificially designed enzymes is available for designing Octopuzymes, they could in theory mimic all protein enzyme reactions on our planet. Moreover, besides L-amino acids, peptides can contain D-amino acids, non-natural amino acids, chemically modified amino acids, nucleotides, vitamins, and manmade catalysts, leading to a huge expansion of catalytic space compared with extant protein enzymes. Once a reaction catalyzed by an Octopuzyme is defined, it could be rapidly evolvable via multiple amino acid substitutions on the eight peptides of Octopuzymes.

Particle‐based MR modeling with diffusion, microstructure, and enzymatic reactions

AbstractPurposeTo develop a novel particle‐based in silico MR model and demonstrate applications of this model to signal mechanisms which are affected by the spatial organization of particles, including metabolic reaction kinetics, microstructural effects on diffusion, and radiofrequency (RF) refocusing effects in gradient‐echo sequences.MethodsThe model was developed by integrating a forward solution of the Bloch equations with a Brownian dynamics simulator. Simulation configurations were then designed to model MR signal dynamics of interest, with a primary focus on hyperpolarized 13C MRI methods. Phantom scans and spectrophotometric assays were conducted to validate model results in vitro.ResultsThe model accurately reproduced the reaction kinetics of enzyme‐mediated conversion of pyruvate to lactate. When varying proportions of restrictive structure were added to the reaction volume, nonlinear changes in the reaction rate measured in vitro were replicated in silico. Modeling of RF refocusing effects characterized the degree of diffusion‐weighted contribution from preserved residual magnetization in nonspoiled gradient‐echo sequences.ConclusionsThese results show accurate reproduction of a range of MR signal mechanisms, establishing the model's capability to investigate the multifactorial signal dynamics such as those underlying hyperpolarized 13C MRI data.

Oxaloacetate anaplerosis differently contributes to pathogenicity in plant pathogenic fungi Fusarium graminearum and F. oxysporum.

Anaplerosis refers to enzymatic reactions or pathways replenishing metabolic intermediates in the tricarboxylic acid (TCA) cycle. Pyruvate carboxylase (PYC) plays an important anaplerotic role by catalyzing pyruvate carboxylation, forming oxaloacetate. Although PYC orthologs are well conserved in prokaryotes and eukaryotes, their pathobiological functions in filamentous pathogenic fungi have yet to be fully understood. Here, we delve into the molecular functions of the ortholog gene PYC1 in Fusarium graminearum and F. oxysporum, prominent fungal plant pathogens with distinct pathosystems, demonstrating variations in carbon metabolism for pathogenesis. Surprisingly, the PYC1 deletion mutant of F. oxysporum exhibited pleiotropic defects in hyphal growth, conidiation, and virulence, unlike F. graminearum, where PYC1 deletion did not significantly impact virulence. To further explore the species-specific effects of PYC1 deletion on pathogenicity, we conducted comprehensive metabolic profiling. Despite shared metabolic changes, distinct reprogramming in central carbon and nitrogen metabolism was identified. Specifically, alpha-ketoglutarate, a key link between the TCA cycle and amino acid metabolism, showed significant down-regulation exclusively in the PYC1 deletion mutant of F. oxysporum. The metabolic response associated with pathogenicity was notably characterized by S-methyl-5-thioadenosine and S-adenosyl-L-methionine. This research sheds light on how PYC1-mediated anaplerosis affects fungal metabolism and reveals species-specific variations, exemplified in F. graminearum and F. oxysporum.