Metabolic Reactions Research Articles

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography-mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected.

Supplemental effects of different production methods of pine needle additives on growth performance, intestinal environment, meat quality and serum of broiler chickens.

Pine needles are rich in many nutrients and exhibit antibacterial and antioxidant biological activities; however, the effects of different production methods of pine needle additives on the growth performance and intestinal flora of broiler chickens are not known. Normal diets were supplemented with pine needle fermentation juice (PNF), pine needle soaking juice (PNS), or pine needle powder (PNP), and the associated effects on growth performance, relative organ weights, intestinal development, intestinal histological morphology, intestinal flora, meat quality, and serum indicators in broiler chickens were observed. The results showed that PNF, PNS, and PNP all significantly improved feed utilisation and promoted the growth and development of broilers. All three additives also significantly improved the structure of the intestinal flora, specifically increasing the diversity of bacteria; increasing the abundance of beneficial bacteria, such as Faecalibacterium, Rikenella, and Blautia; and decreasing the abundance of harmful bacteria, such as Staphylococcus. The antioxidant properties of pine needles were also found to intensify lipid metabolic reactions in the blood, thus leading to lower triglycerides and total cholesterol. Meanwhile, high doses of PNF reduced jejunum and ileum weights and also increased meat yellowness. Lastly, none of PNF, PNS, or PNP had an effect on relative organ weights or intestinal histological morphology. The addition of pine needles to the diet of broiler chickens can effectively promote their growth performance as well as improve their intestinal flora and serum status without side effects; in particular, the dose of 0.2% of either PNF and PNS is expected to have the capacity to replace growth-promoting antibiotics as diet additives.

Identification of New Hepatic Metabolites of Miconazole by Biological and Electrochemical Methods Using Ultra-High-Performance Liquid Chromatography Combined with High-Resolution Mass Spectrometry.

The main objective of this study was to investigate the metabolism of miconazole, an azole antifungal drug. Miconazole was subjected to incubation with human liver microsomes (HLM) to mimic phase I metabolism reactions for the first time. Employing a combination of an HLM assay and UHPLC-HRMS analysis enabled the identification of seven metabolites of miconazole, undescribed so far. Throughout the incubation with HLM, miconazole underwent biotransformation reactions including hydroxylation of the benzene ring and oxidation of the imidazole moiety, along with its subsequent degradation. Additionally, based on the obtained results, screen-printed electrodes (SPEs) were optimized to simulate the same biotransformation reactions, by the use of a simple, fast, and cheap electrochemical method. The potential toxicity of the identified metabolites was assessed using various in silico models.

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs)

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs’ mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

Abstract PO5-08-06: Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Simulation of microbial transport and petroleum hydrocarbon compound biodegradation in a fracture–matrix coupled system

In-situ bioremediation technology is considered an ideal and environment-friendly approach for the largescale treatment of petroleum-contaminated soil. Most earlier studies on bioremediation have focused on the biostimulation techniques, which can enhance the biodegradation capabilities of indigenous bacteria, rather than on bioaugmentation involving the introduction of exogenous microorganisms. Additionally, contaminated soil sites typically contain both natural and artificial fractures. These fractures serve as preferential transport pathways, complicating the transport of bacteria and related solutes. This study proposes a mathematical model to simulate the transport of exogenous bacteria and electron acceptors during the degradation of petroleum hydrocarbon pollutants in a fractured aquifer system. The model incorporates the processes such as microbial convection, diffusion, adsorption, chemotaxis, growth, death, and microbial metabolic reactions. Simulation results demonstrate that considering microbial transport significantly affects the prediction of contaminant degradation, particularly with regard to the transport of electron acceptors. The growth rate of bacteria has an evident effect on the efficiency of contaminant remediation, and microbial chemotaxis can significantly enhance the degradation efficiency. The proposed mathematical model serves as a valuable tool for assessing the effectiveness of bioaugmentation strategies and can aid in the design and optimization of remediation efforts for contaminated and fractured aquifers.

Glutathione: a key modulator of plant defence and metabolism through multiple mechanisms.

Redox reactions are fundamental to energy conversion in living cells, and also determine and tune responses to the environment. Within this context, the tripeptide glutathione plays numerous roles. As an important antioxidant, glutathione confers redox stability on the cell and also acts as an interface between signalling pathways and metabolic reactions that fuel growth and development. It also contributes to the assembly of cell components, biosynthesis of sulfur-containing metabolites, inactivation of potentially deleterious compounds, and control of hormonal signalling intensity. The multiplicity of these roles probably explains why glutathione status has been implicated in influencing plant responses to many different conditions. In particular, there is now a considerable body of evidence showing that glutathione is a crucial player in governing the outcome of biotic stresses. This review provides an overview of glutathione synthesis, transport, degradation, and redox turnover in plants. It examines the expression of genes associated with these processes during pathogen challenge and related conditions, and considers the diversity of mechanisms by which glutathione can influence protein function and gene expression.

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae.

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. This study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

The genetic landscape of a metabolic interaction

While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focus on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We use deep mutational scanning to quantify the growth rate effect of 2696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

Living Artificial Skin: Photosensitizer and Cell Sandwiched Bacterial Cellulose for Chronic Wound Healing.

Chronic wounds pose a significant global public health challenge due to their suboptimal treatment efficacy caused by bacterial infections and microcirculatory disturbances. Inspired by the biofunctionality of natural skin, an artificial skin (HV@BC@TBG) is bioengineered with bacterial cellulose (BC) sandwiched between photosensitizers (PS) and functionalized living cells. Glucose-modified PS (TBG) and vascular endothelial growth factor (VEGF)-functionalized living cells (HV) are successively modified on each side of BC through biological metabolism and bio-orthogonal reaction. As the outermost layer, the TBG layer can generate reactive oxygen species (ROS) upon light illumination to efficiently combat bacterial infections. The HV layer is the inner layer near the diabetic wound, which servs as a living factory to continuously secrete VEGF to accelerate wound repair by promoting fibroblast proliferation and angiogenesis. The sandwiched structural artificial skin HV@BC@TBG is nontoxic, biocompatible, and demonstrated its ability to significantly accelerate the healing process of infected diabetic wounds, rendering it a promising next-generation medical therapy for chronic wound management.

Isotopically nonstationary metabolic flux analysis of plants: recent progress and future opportunities.

Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST-MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast-growing algae, and produced a synergistic multi-organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST-MFA to correct an oversimplified understanding of plant metabolism.

One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs aretrapped in microfluidic devices for analysis by optical microscopy.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

Deciphering Microbiome, Transcriptome, and Metabolic Interactions in the Presence of Probiotic Lactobacillus acidophilus against Salmonella Typhimurium in a Murine Model.

Salmonella enterica serovar Typhimurium (S. Typhimurium), a foodborne pathogen that poses significant public health risks to humans and animals, presents a formidable challenge due to its antibiotic resistance. This study explores the potential of Lactobacillus acidophilus (L. acidophilus 1.3251) probiotics as an alternative strategy to combat antibiotic resistance associated with S. Typhimurium infection. In this investigation, twenty-four BALB/c mice were assigned to four groups: a non-infected, non-treated group (CNG); an infected, non-treated group (CPG); a group fed with L. acidophilus but not infected (LAG); and a group fed with L. acidophilus and challenged with Salmonella (LAST). The results revealed a reduction in Salmonella levels in the feces of mice, along with restored weight and improved overall health in the LAST compared to the CPG. The feeding of L. acidophilus was found to downregulate pro-inflammatory cytokine mRNA induced by Salmonella while upregulating anti-inflammatory cytokines. Additionally, it influenced the expression of mRNA transcript, encoding tight junction protein, oxidative stress-induced enzymes, and apoptosis-related mRNA expression. Furthermore, the LEfSe analysis demonstrated a significant shift in the abundance of critical commensal genera in the LAST, essential for maintaining gut homeostasis, metabolic reactions, anti-inflammatory responses, and butyrate production. Transcriptomic analysis revealed 2173 upregulated and 506 downregulated differentially expressed genes (DEGs) in the LAST vs. the CPG. Functional analysis of these DEGs highlighted their involvement in immunity, metabolism, and cellular development. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis indicated their role in tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), chemokine, Forkhead box O (FOXO), and transforming growth factor (TGF-β) signaling pathway. Moreover, the fecal metabolomic analysis identified 929 differential metabolites, with enrichment observed in valine, leucine, isoleucine, taurine, glycine, and other metabolites. These findings suggest that supplementation with L. acidophilus promotes the growth of beneficial commensal genera while mitigating Salmonella-induced intestinal disruption by modulating immunity, gut homeostasis, gut barrier integrity, and metabolism.

Impaired coenzyme A homeostasis in cardiac dysfunction and benefits of boosting coenzyme A production with vitamin B5 and its derivatives in the management of heart failure.

Coenzyme A (CoA) is an essential cofactor required for over a hundred metabolic reactions in the human body. This cofactor is synthesized de novo in our cells from vitamin B5, also known as pantothenic acid, a water-soluble vitamin abundantly present in vegetables and animal-based foods. Neurodegenerative disorders, cancer, and infectious diseases have been linked to defects in de novo CoA biosynthesis or reduced levels of this coenzyme. There is now accumulating evidence that CoA limitation is a critical pathomechanism in cardiac dysfunction too. In the current review, we will summarize our current knowledge on CoA and heart failure, with emphasis on two primary cardiomyopathies, phosphopantothenoylcysteine synthetase and phosphopantothenoylcysteine decarboxylase deficiency disorders biochemically characterized by a decreased level of CoA in patients' samples. Hence, we will discuss the potential benefits of CoA restoration in these diseases and, more generally, in heart failure, by vitamin B5 and its derivatives pantethine and 4'-phosphopantetheine.

The Metabolism of Coenzyme A and Its Derivatives Plays a Crucial Role in Diseases.

Coenzyme A (CoA) functions as a crucial carrier of acyl groups within cells, playing a fundamental role in regulating acyl transfer reactions and participating in cellular metabolic processes. As the principal substrate and cofactor engaged in diverse metabolic reactions, CoA and its derivatives exert central influence over various physiological processes, primarily modulating lipid and ketone metabolism, as well as protein modification. This paper presents a comprehensive review of the molecular mechanisms by which CoA influences the onset and progression of cancer, cardiovascular disease (CVD), neurodegenerative disorders, and other illnesses. The main focal points include the following. (1) In cancer, enzymes such as acetyl-CoA synthetase 2, ATP citrate lyase, and acetyl-CoA carboxylase regulate lipid synthesis and energy metabolism by modulating acetyl-CoA levels. (2) In CVD, the effects of enzymes such as stearoyl-CoA desaturase-1, 3-hydroxy-3-methylglutaryl-CoA (HMGC) synthase 2, and HMGC reductase on the formation and advancement of these diseases are elucidated by their regulation of CoA metabolism across multiple organs. (3) In neurodegenerative disorders, the significance of CoA in maintaining cholesterol homeostasis in the brain and its implications on the development of such disorders are thoroughly discussed. The metabolic processes involving CoA and its derivatives span all physiological aspects within cells, playing a critical role in the onset and progression of various diseases. Elucidating the role of CoA in these conditions yields important insights that can serve as valuable references and guidance for disease diagnosis, treatment, and drug development.

Functional characterization, structural basis, and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2′-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2′-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pKa calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2′-O-glycosylation (kcat/Km, 0.23 L/(s·μmol), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 μM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Interactions between Epichloë endophyte and the plant microbiome impact nitrogen responses in host Achnatherum inebrians plants.

The clavicipitaceous fungus Epichloë gansuensis forms symbiotic associations with drunken horse grass (Achnatherum inebrians), providing biotic and abiotic stress protection to its host. However, it is unclear how E. gansuensis affects the assembly of host plant-associated bacterial communities after ammonium nitrogen (NH4+-N) treatment. We examined the shoot- and root-associated bacterial microbiota and root metabolites of A. inebrians when infected (I) or uninfected (F) with E. gansuensis endophyte. The results showed more pronounced NH4+-N-induced microbial and metabolic changes in the endophyte-infected plants compared to the endophyte-free plants. E. gansuensis significantly altered bacterial community composition and β-diversity in shoots and roots and increased bacterial α-diversity under NH4+-N treatment. The relative abundance of 117 and 157 root metabolites significantly changed with E. gansuensis infection under water and NH4+-N treatment compared to endophyte-free plants. Root bacterial community composition was significantly related to the abundance of the top 30 metabolites [variable importance in the projection (VIP) > 2 and VIP > 3] contributing to differences between I and F plants, especially alkaloids. The correlation network between root microbiome and metabolites was complex. Microorganisms in the Proteobacteria and Firmicutes phyla were significantly associated with the R00693 metabolic reaction of cysteine and methionine metabolism. Co-metabolism network analysis revealed common metabolites between host plants and microorganisms.IMPORTANCEOur results suggest that the effect of endophyte infection is sensitive to nitrogen availability. Endophyte symbiosis altered the composition of shoot and root bacterial communities, increasing bacterial diversity. There was also a change in the class and relative abundance of metabolites. We found a complex co-occurrence network between root microorganisms and metabolites, with some metabolites shared between the host plant and its microbiome. The precise ecological function of the metabolites produced in response to endophyte infection remains unknown. However, some of these compounds may facilitate plant-microbe symbiosis by increasing the uptake of beneficial soil bacteria into plant tissues. Overall, these findings advance our understanding of the interactions between the microbiome, metabolome, and endophyte symbiosis in grasses. The results provide critical insight into the mechanisms by which the plant microbiome responds to nutrient stress in the presence of fungal endophytes.