Enzyme In Pathway Research Articles

Decoding eggplant fruit: Multi-omics profiling of caffeoyl-CoA-3-OMT expression

Eggplant (Solanum melongena L.) is a rich source of health-promoting phenolic acids, mainly chlorogenic acid (CGA), and trace elements. CGA has been shown to have antioxidant, anti-inflammatory, neuroprotective, cardioprotective, anticancer, and antidiabetic properties. The natural genome of eggplants contains essential genes for beneficial traits like drought tolerance, disease resistance, and high concentrations of medicinal substances. Transcriptomic analysis of eggplant overexpressing the hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene revealed upregulation of 2165 genes, including three similar to HQT and cinnamate-4-hydroxylase (C4H). Sequence similarity analysis showed homology to caffeoyl-CoA O-methyltransferases, key enzymes in the phenylpropanoid pathway leading to CGA biosynthesis. Gene ontology and KEGG pathway analysis identified 5 highly upregulated glycosyltransferase family 43 genes (650.3-fold change). Glycosyltransferases like UDP-glucose:cinnamate glucosyltransferase and cinnamate-glucose 4′-O-glucosyltransferase are crucial for CGA production in eggplant. qRT-PCR validated the potential upregulation of HQT and C4H in transgenic eggplants. Comparative analysis revealed eggplant has the highest CGA content (5–8.1 g/kg dry weight) among vegetables, with foliar CGA acting as a natural insecticide. CGA offers antioxidant, hypoglycemic, antiviral, and hepatoprotective benefits to humans. This study highlights the role of HQT and C4H in elevating the medicinal properties of eggplant through CGA biosynthesis pathway engineering.

Impact of nitrogen rates on biosynthesis pathways: A comparative study of diterpene synthases in clerodane diterpenoids and enzymes in benzylisoquinoline alkaloids

Tinospora sagittata is rich in secondary metabolites used in traditional medicine. However, environmental factors impact key enzymes in metabolite synthesis, highlighting the need for improved growth conditions. This study employs transcriptomics and metabolomics to assess nitrogen's impact on enzymes in secondary metabolites biosynthesis pathways. The gene expressions of berberine bridge enzymes (BBEs) like TsBBE2 had peak expression in low nitrogen treatments (A0 and A1) but were absent in higher nitrogen treatments (A2 and A3). Similar trends were observed for other enzymes such as (S)-scoulerine 9-O-methyltransferase (TsCMT3), Tetrahydroberberine oxidase (TsSTOX), and Columbamine O-methyltransferase (TsCoCOMT2-4) in response to nitrogen levels. In examining gene families related to diterpene synthases (diTPS), 1-deoxyxylulose 5-phosphate synthase (TsDXR1) expression increased with higher nitrogen fertilizer, while TsDXR2 peaked at maximal nitrogen levels. Geranylgeranyl diphosphate synthase (TsGGPP3 and TsGGPP5) decreased with nitrogen levels. (−)-kolavenyl diphosphate synthase (KPS) genes had higher expression in treatments, while ent-kaurene synthase (KSL) genes, especially TsKSL1 and TsKSL2, showed higher expression in control conditions with lower nitrogen fertilizer. Metabolite analysis confirmed more upregulated compounds in A3 compared to A0. These findings have practical implications for agriculture and pharmaceuticals, highlighting the link between nitrogen fertilization and specialized metabolism in medicinal plants.

Cobalt-modified digestate-derived biochar enhances kitchen waste anaerobic dry digestion: Performance, microbial mechanisms, and metabolic pathways

To achieve simultaneous in-situ high-value utilization of digestate and efficient resource recovery of kitchen waste (KW). In this work, Co-modified digestate-derived biochar (DDB-Co) was prepared for the first time, the effects of DDB-Co on KW mesophilic dry anaerobic digestion were investigated, and the underlying mechanisms were elucidated. The results showed that the specific surface area of DDB-Co increased to 98.7 m2/g, which was 37.4 % higher than that of the unmodified group (DDB). DDB-Co exhibited a dose-dependent effect on biogas production from the KW dry digestion, with the optimal dose of 4.0 g/L (calculated on a solid content) DDB-Co yielding 205.3 mL/(g·VS) of biogas, representing an 80.9 % increase compared to the CK group. However, higher doses, such as 8.0 g/L DDB-Co, reduced the cumulative biogas production. Mechanistic analysis indicated that 4.0 g/L DDB-Co facilitated the dissolution of particulate organic matter and accelerates the conversion process of volatile fatty acids (VFA) in KW, thereby preventing excessive acidification. Microbial community structure analysis revealed that DDB-Co reduced microbial richness and diversity but selectively enriched microorganisms such as Lactobacillus and Methanothrix, accelerating the direct interspecific electron transfer (DIET) reaction rate and increasing biogas production. Metagenomic analysis demonstrated that DDB-Co primarily upregulated the relative abundances of key enzymes in the acetate decarboxylation methanogenesis pathway, such as acetate kinase (ackA), phosphate acetyltransferase (pta), and acetyl-CoA synthetase (acs), enhancing biogas yield and maintaining system stability. The findings of this work expand the high-value utilization pathways for digestate and provide an alternative solution for the efficient resource recovery of KW.

Myocardial injury and heart failure biomarkers in acute intermittent porphyria

Abstract Background Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by the defect in the hydroxymethylbilane synthase gene, encoding the third enzyme in heme biosynthesis pathway. The main symptoms include abdominal pain, vomiting, paralysis, confusion. Exacerbation of AIP may manifest with elevation of blood pressure and tachycardia due to hyperactivity of sympathetic nervous system. Data regarding cardiac function in AIP are scarce. Purpose The aim of this study was to assess the myocardial injury and heart failure biomarkers during AIP exacerbations and remission. Methods This cohort prospective study encompassed patients at the age 18-65 years who had at least one exacerbation of AIP in their lifetime. Patients examined during the AIP exacerbations were reassessed in the remission phase. The concentrations of sodium, troponin T, CK-MB mass, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and metoxycatecholamines in blood were assessed. Blood pressure was measured with 24-hour ABPM. Echocardiography was performed according to the current recommendations. Results The total number of 70 patients with AIP were enrolled to the study (April 2019 - March 2023). We assessed 36 patients in both phases of disease, mean age 37 years (21-63), 75% females. The median of prior AIP exacerbations was 8. During the exacerbations a significant increase in the concentrations of NT-proBNP (250 [97 ; 501] vs. 89 [48 ; 140] pg/mL, p<0.001), metanephrine (49.0 [37.8 ; 61.2] vs. 41.4 [36.8 ; 53.3] pg/mL, p=0.009) and normetanephrine (169.8 [118.2 ; 327.2] vs. 93.2 [78.1 ; 120.9] pg/mL, p<0.001) was observed. Conversely, sodium concentrations decreased (136 [133 ; 140] vs. 140 [139 ; 142] mmol/L, p<0.001). There were no significant differences in the values of troponin (5.4 [4.0 ; 11.3] vs. 5.6 [3.7 ; 9.5] ng/L, p=0.445), CK-MB mass (1.88 [0.84 ; 2.64] vs. 1.21 [0.83 ; 2.80] ng/mL, p=0.80). In the acute phases of AIP the levels of NT-proBNP were correlated negatively with the values of sodium concentrations (r=-0.36, p=0.032) and positively with average systolic blood pressure (r=0.32, p=0.06). Echocardiography showed an increase in left ventricular (LV) mass indices in some patients hospitalized due to AIP symptoms. Sodium concentrations correlated negatively with these changes (r=-0.36, p=0.032). LV ejection fraction was slightly higher during AIP exacerbations. There were no significant differences in other parameters of LV systolic and diastolic function. Conclusions During acute intermittent porphyria (AIP) exacerbations there is an increase in the concentrations of NT-proBNP and catecholamines in blood. In most patients the levels of markers of myocardial injury, such as troponin and CK-MB mass, do not differ significantly between phases of disease. During AIP exacerbations some patients experience an increase in LV mass which may be associated with hyponatremia. In most cases cardiac function do not change significantly.Frequency of abnormal laboratory resultsResults of echocardiography

Genome-wide identification and expression analysis of the OSC gene family in Platycodon grandiflorus

Platycodon grandiflorus stands as one of the most extensively utilized traditional Chinese medicinal herbs, with triterpenoids and their derivatives serving as its primary medicinal components. Oxido squalene cyclase (OSC), serving as a crucial enzyme in the triterpenoid synthesis pathway, has the capability to enzymatically generate significant quantities of sterols and triterpenoid intermediates. While the OSC gene family has been identified in numerous species, bioinformatics research on this family remains scant. Presently, the specific members of this gene family in Platycodon grandiflorus have yet to be definitively determined. In this study, we successfully identified a total of 15 PgOSC genes within the genome of Platycodon grandiflorus by conducting homology comparisons. These genes were discovered to be unevenly distributed across the five chromosomes of the species, organized in the form of gene clusters. Subsequently, we conducted a thorough analysis of the OSC gene family’s evolutionary relationship by constructing a phylogenetic tree. Other characteristics of PgOSC family members, including gene structure, conserved motifs, protein three-dimensional structure, subcellular localization, and cis-acting elements were thoroughly characterized. Furthermore, We analyzed the expression of PgOSC gene in different tissues of Platycodon grandiflorus by qRT-PCR, and found that the expression of PgOSC genes in root was higher than that in stem and leaf. Upon comparing the effects of salt, heat, and drought treatments, we observed a significant induction of PgOSC gene expression in Platycodon grandiflorus specifically under salt stress conditions. In summary, this study comprehensively identified and analyzed the OSC gene family, aiming to provide basic biological information for exploring the members of PgOSC gene family.

A simplified liquid chromatography-mass spectrometry methodology to probe the shikimate and aromatic amino acid biosynthetic pathways in plants.

Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback-insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post-chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post-chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants.

Time-Course Transcriptome Analysis Reveals Distinct Transcriptional Regulatory Networks in Resistant and Susceptible Grapevine Genotypes in Response to White Rot

Grapevine (Vitis vinifera L.) is a globally significant economic crop. However, its widely cultivated varieties are highly susceptible to white rot disease. To elucidate the mechanisms of resistance in grapevine against this disease, we utilized time-ordered gene co-expression network (TO-GCN) analysis to investigate the molecular responses in the grapevine varieties ‘Guifeimeigui’ (GF) and ‘Red Globe’ (RG). An assessment of their resistance demonstrated that GF is highly resistant to white rot, whereas RG is highly susceptible. We conducted transcriptome sequencing and a TO-GCN analysis on leaf samples from GF and RG at seven time points post-infection. Although a significant portion of the differentially expressed genes related to disease resistance were shared between GF and RG, the GF variety rapidly activated its defense mechanisms through the regulation of transcription factors during the early stages of infection. Notably, the gene VvLOX3, which is a key enzyme in the jasmonic acid biosynthetic pathway, was significantly upregulated in GF. Its upstream regulator, Vitvi08g01752, encoding a HD-ZIP family transcription factor, was identified through TO-GCN and yeast one-hybrid analyses. This study provides new molecular insights into the mechanisms of grapevine disease resistance and offers a foundation for breeding strategies aimed at enhancing resistance.

Bempedoic Acid, the First-in-Class Oral ATP Citrate Lyase Inhibitor with Hypocholesterolemic Activity: Clinical Pharmacology and Drug–Drug Interactions

Bempedoic acid is a new drug that improves the control of cholesterol levels, either as monotherapy or in combination with existing lipid-lowering therapies, and shows clinical efficacy in cardiovascular disease patients. Thus, patients with comorbidities and under multiple therapies may be eligible for bempedoic acid, thus facing the potential problem of drug–drug interactions (DDIs). Bempedoic acid is a prodrug administered orally at a fixed daily dose of 180 mg. The dicarboxylic acid is enzymatically activated by conjugation with coenzyme A (CoA) to form the pharmacologically active thioester (bempedoic acid–CoA). This process is catalyzed by very-long-chain acyl-CoA synthetase 1 (ACSVL1), expressed almost exclusively at the hepatic level. Bempedoic acid–CoA is a potent and selective inhibitor of ATP citrate lyase (ACL), a key enzyme in the biosynthetic pathway of cholesterol and fatty acids. The drug reduces low-density lipoprotein–cholesterol (LDL-C) (20–25%), non-high-density lipoprotein–cholesterol (HDL-C) (19%), apolipoprotein B (apoB) (15%), and total cholesterol (16%) in patients with hypercholesterolemia or mixed dyslipidemia. The drug has a favorable pharmacokinetics profile. Bempedoic acid and its metabolites are not substrates or inhibitors/inducers of cytochrome P450 (CYP450) involved in drug metabolism. On the other hand, bempedoic acid–glucuronide is a substrate for organic anion transporter 3 (OAT3). Bempedoic acid and its glucuronide are weak inhibitors of the OAT2, OAT3, and organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3). Thus, bempedoic acid could inhibit (perpetrator) the hepatic uptake of OATP1B1/3 substrate drugs and the renal elimination of OAT2 and OAT3 substrates and could suffer (victim) the effect of OAT3 transporter inhibitors, reducing its renal elimination. Based on these pharmacological characteristics, here, we describe the potential DDIs of bempedoic acid with concomitant medications and the possible clinical implications.

Whole genome sequencing of Castanea mollissima and molecular mechanisms of sugar and starch synthesis

The chestnut tree exhibits self-incompatibility, where the selection of the male parent (pollen xenia) significantly affects seed starch metabolism, as well as fruit yield and quality. Despite its importance, the molecular mechanisms underlying pollen xenia remains largely unknown. In this study, we utilized the ‘Lan You’ variety of C. mollissima to construct a high-quality reference genome. As a result, a first Telomere-to-telomere (T2T) gap-free genome for this species was successfully assembled. A total of 560 transcription factors and 22 structural genes were identified as consistent across the TO-GCNs, indicating a consistent regulation pattern in the co-expression of genes involved in starch accumulation. These networks were further divided into three sub-networks: T1, T2, and T3. Among these, the T1 and T2 sub-networks exhibited a higher number of structural genes with consistent regulation patterns and were closely associated with sugar biosynthesis. The gene SBE (Camol08G0254600) was identified as the hub gene with the highest degree of connectivity, encoding a key rate-limiting enzyme in the amylopectin biosynthesis pathway. This study provides a foundation for further research on C. mollissima population genetics, genetic improvement, and strategies aimed at enhancing yield and quality.

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP3 [inositol(1, 4, 5)trisphosphate], IP4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

Sulfur metabolism under stress: Oxidized glutathione inhibits methionine biosynthesis by destabilizing the enzyme cystathionine γ-synthase.

Cysteine is the precursor for the biosynthesis of glutathione, a key stress-protective metabolite, and methionine, which is imperative for cell growth and protein synthesis. The exact mechanism that governs the routing of cysteine toward glutathione or methionine during stresses remains unclear. Our study reveals that under oxidative stress, methionine and glutathione compete for cysteine and that the increased oxidized glutathione (GSSG) levels under stress hinder methionine biosynthesis. Moreover, we find that inhibition occurs as GSSG binds to and accelerates the degradation of cystathionine γ-synthase, a key enzyme in the methionine synthesis pathway. Consequently, this leads to a reduction in the flux toward methionine-derived metabolites and redirects cysteine utilization toward glutathione, thereby enhancing plant protection. Our study suggests a novel regulatory feedback loop involving glutathione, methionine, and cysteine, shedding light on the plant stress response and the adaptive rerouting of cysteine. These findings offer new insights into the intricate balance of growth and protection in plants and its impact on their nutritional value due to low methionine levels under stress.

Genetically encoded green fluorescent sensor for probing sulfate transport activity of solute carrier family 26 member a2 (Slc26a2) protein

Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers.

The Biosynthesis and Applications of Protein Lipidation.

Protein lipidation dramatically affects protein structure, localization, and trafficking via remodeling protein-membrane and protein-protein interactions through hydrophobic lipid moieties. Understanding the biosynthesis of lipidated proteins, whether natural ones or mimetics, is crucial for reconstructing, validating, and studying the molecular mechanisms and biological functions of protein lipidation. In this Perspective, we first provide an overview of the natural enzymatic biosynthetic pathways of protein lipidation in mammalian cells, focusing on the enzymatic machineries and their chemical linkages. We then discuss strategies to biosynthesize protein lipidation in mammalian cells by engineering modification machineries and substrates. Additionally, we explore site-specific protein lipidation biosynthesis in vitro via enzyme-mediated ligations and in vivo primarily through genetic code expansion strategies. We also discuss the use of small molecule tools to modulate the process of protein lipidation biosynthesis. Finally, we provide concluding remarks and discuss future directions for the biosynthesis and applications of protein lipidation.

6-hydroxy-3-succinoylsemialdehyde-pyridine as a potential inhibitor of schizophrenia-associated enzyme

Schizophrenia’s cognitive deficits limit quality of life. Current drugs are ineffective. This study investigates 6-hydroxy-3-succinoylsemialdehyde-pyridine (6H3SAP), a nicotine derivative, as a potential inhibitor of KATII, an enzyme in the kynurenine pathway linked to cognitive dysfunction. Molecular docking was employed to assess the binding affinity of 6H3SAP to the KATII enzyme. The results indicated that 6H3SAP interacted with the KATII active site, demonstrating a binding affinity comparable to nicotine (previously suggested as a potential KATII blocker) but lower than NS1502, a well-established KATII inhibitor. Subsequent molecular dynamics simulations provided further insights into the interaction details between 6H3SAP and KATII. These simulations revealed the formation of stable interactions between 6H3SAP and key amino acid residues within the KATII binding pocket. Though promising, further research is needed to assess 6H3SAP’s efficacy and safety. Surprisingly, the molecular dynamics data also proposed that the nicotine-KATII tie is weak.

Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis.

KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations.

Molecular insights and functional analysis of isocitrate dehydrogenase in two gram-negative pathogenic bacteria.

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70kDa. They are completely dependent on the coenzyme NADP+ and are inactive towards NAD+. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn2+ and at pH 7.8 in the presence of Mg2+. Its optimal catalytic performance is achieved with both ions at 55°C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn2+ and Mg2+, respectively, and exhibits optimal catalytic performance at 45°C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45°C for 20min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP+ to NAD+ by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies.

The reverse transsulfuration pathway affects the colonic microbiota and contributes to colitis in mice

Cystathionine γ-lyase (CTH) is a critical enzyme in the reverse transsulfuration pathway, the major route for the metabolism of sulfur-containing amino acids, notably converting cystathionine to cysteine. We reported that CTH supports gastritis induced by the pathogen Helicobacter pylori. Herein our aim was to investigate the role of CTH in colonic inflammation. First, we found that CTH is induced in the colon mucosa in mice with dextran sulfate sodium-induced colitis. Expression of CTH was completely absent in the colon of Cth–/– mice. We observed that clinical and histological parameters are ameliorated in Cth-deficient mice compared to wild-type animals. However, Cth deletion had no effect on tumorigenesis and the level of dysplasia in mice treated with azoxymethane-DSS, as a reliable model of colitis-associated carcinogenesis. Mechanistically, we determined that the deletion of the gene Slc7a11 encoding for solute carrier family 7 member 11, the transporter of the anionic form of cysteine, does not affect DSS colitis. Lastly, we found that the richness and diversity of the fecal microbiota were significantly increased in Cth–/– mice compared to both WT and Slc7a11–/– mice. In conclusion, our data suggest that the enzyme CTH represents a target for clinical intervention in patients with inflammatory bowel disease, potentially by beneficially reshaping the composition of the gut microbiota.

Gene expression of kynurenine pathway enzymes in depression and following electroconvulsive therapy.

This study aimed to investigate changes in mRNA expression of the kynurenine pathway (KP) enzymes tryptophan 2, 3-dioxygenase (TDO), indoleamine 2, 3-dioxygenase 1 and 2 (IDO1, IDO2), kynurenine aminotransferase 1 and 2 (KAT1, KAT2), kynurenine monooxygenase (KMO) and kynureninase (KYNU) in medicated patients with depression (n = 74) compared to age- and sex-matched healthy controls (n = 55) and in patients with depression after electroconvulsive therapy (ECT). Associations with mood score (24-item Hamilton Depression Rating Scale, HAM-D24), plasma KP metabolites and selected glucocorticoid and inflammatory immune markers known to regulate KP enzyme expression were also explored. HAM-D24 was used to evaluate depression severity. Whole blood mRNA expression was assessed using quantitative real-time polymerase chain reaction. KAT1, KYNU and IDO2 were significantly reduced in patient samples compared to control samples, though results did not survive statistical adjustment for covariates or multiple comparisons. ECT did not alter KP enzyme mRNA expression. Changes in IDO1 and KMO and change in HAM-D24 score post-ECT were negatively correlated in subgroups of patients with unipolar depression (IDO1 only), psychotic depression and ECT responders and remitters. Further exploratory correlative analyses revealed altered association patterns between KP enzyme expression, KP metabolites, NR3C1 and IL-6 in depressed patients pre- and post-ECT. Further studies are warranted to determine if KP measures have sufficient sensitivity, specificity and predictive value to be integrated into stress and immune associated biomarker panels to aid patient stratification at diagnosis and in predicting treatment response to antidepressant therapy.

Fungal L-Methionine Biosynthesis Pathway Enzymes and Their Applications in Various Scientific and Commercial Fields.

L-methionine (L-Met) is one of the nine proteinogenic amino acids essential for humans since, in human cells, there are no complete pathways for its biosynthesis from simple precursors. L-Met plays a crucial role in cellular function as it is required for proper protein synthesis, acting as an initiator. Additionally, this amino acid participates in various metabolic processes and serves as a precursor for the synthesis of S-adenosylmethionine (AdoMet), which is involved in the methylation of DNA molecules and phospholipids, as well as in maintaining genome stability. Due to its importance, fungal L-methionine biosynthesis pathway enzymes are being intensively studied. This review presents the current state of the art in terms of their cellular function, usefulness as molecular markers, antifungal targets, or industrial approaches.

A scaffold protein manages the biosynthesis of steroidal defense metabolites in plants.

Solanaceae plants produce two major classes of valuable sterol derived natural products-steroidal glycoalkaloids and steroidal saponins-from a common cholesterol precursor. Attempts to heterologously produce these molecules have consistently failed, although the genes responsible for each biosynthetic step have been identified. Here we identify a cellulose synthase like protein, an unexpected biosynthetic component that interacts with the early pathway enzymes, enabling steroidal scaffolds production in plants. Moreover, knockout of this gene in black nightshade, Solanum nigrum resulted in plants lacking both steroidal alkaloids and saponins. Unexpectedly, these knockout plants also revealed that steroidal saponins deter serious agricultural insect pests. This discovery provides the missing link to engineer these high value steroidal molecules, and also pinpoints the ecological role for the steroidal saponins.