Search Results (37)

The effect of salt stress (150 mM NaCl) on the expression of genes, methylation of their promoters, and enzymatic activity of glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), and the 2-oxoglutarate (2-OG)–dehydrogenase (2-OGDH) complex was studied in maize (Zea mays L.). GDH activity increased continuously under salt stress, being 3-fold higher after 24 h. This was accompanied by the appearance of a second isoform with lower electrophoretic mobility. The expression of the Gdh1 gene strongly increased after 6–12 h of incubation, which corresponded to the demethylation of its promoter, while Gdh2 gene expression slightly increased after 2–6 h and then decreased. GAD activity gradually increased in the first 12 h, and then returned to the control level. This corresponded to the increase of Gad expression and its demethylation. Salt stress led to a 2-fold increase in the activity of 2-OGDH during the first 6 h of NaCl treatment, then the activity returned to the control level. Expression of the genes Ogdh1 and Ogdh3 peaked after 1–2 h of incubation. After 6–8 h with NaCl, the expression of these genes declined below the control levels, which correlated with the higher methylation of their promoters. We conclude that salt stress causes a redirection of the 2-OG flux to the γ-aminobutyric acid shunt via its amination to glutamate, by altering the expression of the Gdh1 and Gdh2 genes, which likely promotes the assembly of the native GDH molecule having a different subunit composition and greater affinity for 2-OG. Full article

The aim of the study was to assess the impact of plant extracts from hemp inflorescences (H10—10% and H20—20%), as well as a mixture of extracts from hemp inflorescences, sage, and tansy leaves (M10—10% and M20—20%) on phytotoxicity and selected physiological and biometric parameters of wheat seedlings, as well as the biological activity of soil in a growth chamber experiment. In all experimental combinations, a low phytotoxicity of the extracts was observed in the form of leaf tip yellowing, classified as first-degree damage or its complete absence. The plant extracts and their mixtures, except for the H20 extract, had an inhibitory effect on the development of fungal pathogens, especially Fusarium spp. The H20 extract increased the fresh and dry weight of root seedlings. The tested extracts also had a positive effect on the chlorophyll content in seedlings. The highest chlorophyll concentrations were recorded for the seedlings sprayed with the M20 extract mixture. The applied plant extracts influenced the activity of soil enzymes. The highest activity of catalase and dehydrogenases was observed after spraying seedlings with M20, while the lowest was recorded after applying H10. Of all the tested groups of soil environment compounds included in the Biolog EcoPlates test, carbohydrates and carboxylic acids were most actively utilized. Conversely, amines and amides constituted the group of compounds utilized the least frequently. The present study demonstrated the high effectiveness of plant extracts on wheat seedlings due to their biocidal action against phytopathogenic fungi and increased biological activity of the soil. This research serves as an initial phase of work, which will aim to verify the results obtained under field conditions, as well as assess the biological stability of the extracts. Full article

Meso-diaminopimelate dehydrogenase (meso-DAPDH) from Corynebacterium glutamicum ATCC13032 (CgDAPDH) is a type I meso-DAPDH that shows obvious preference toward meso-diaminopimelate (meso-DAP) and exhibits almost no amination activity toward 2-keto acids. There are seven distinct conserved insertions and deletions (indels) between type I and type II meso-DAPDH. The current functional analysis of indels is not comprehensive in meso-DAPDH. Continuing from our previous work on these indels, we first examined the functions of the other indels shown as insertion residues in type I CgDAPDH. Alanine mutations in M216, T240, K289, and Q290 lost at least 40% of their activity, highlighting the importance of these four sites in CgDAPDH. Molecular dynamic analysis indicated that the four non-active sites altered the dynamic network of interactions within the protein. Subsequently, these four sites together with the previously identified indel-related residues R180, L176, and H193 were targeted by site-saturation mutagenesis to improve the amination ability of CgDAPDH toward pyruvic acid. The most significant improvement was observed with the mutant CgL176R, which showed a six-fold increase toward pyruvic acid in k_cat/K_m compared to wild-type CgDAPDH. Overall, our study provides new hotspots and ideas for the subsequent protein engineering of CgDAPDH, which may also be applied to other meso-DAPDHs. Full article

Our previous study showed that COPPER-CONTAINING AMINE OXIDASE (CuAO) and AMINOALDEHYDE DEHYDROGENASE (AMADH) could regulate the accumulation of γ-aminobutyric acid (GABA) in tea through the polyamine degradation pathway. However, their biological function in drought tolerance has not been determined. In this study, Camellia sinensis (Cs) CsCuAO1 associated with CsAMADH1 conferred drought tolerance, which modulated GABA levels in tea plants. The results showed that exogenous GABA spraying effectively alleviated the drought-induced physical damage. Arabidopsis lines overexpressing CsCuAO1 and CsAMADH1 exhibited enhanced resistance to drought, which promoted the synthesis of GABA and putrescine by stimulating reactive oxygen species’ scavenging capacity and stomatal movement. However, the suppression of CsCuAO1 or CsAMADH1 in tea plants resulted in increased sensitivity to drought treatment. Moreover, co-overexpressing plants increased GABA accumulation both in an Agrobacterium-mediated Nicotiana benthamiana transient assay and transgenic Arabidopsis plants. In addition, a GABA transporter gene, CsGAT1, was identified, whose expression was strongly correlated with GABA accumulation levels in different tissues under drought stress. Taken together, CsCuAO1 and CsAMADH1 were involved in the response to drought stress through a dynamic GABA-putrescine balance. Our data will contribute to the characterization of GABA’s biological functions in response to environmental stresses in plants. Full article

The effective synthesis of N-benzyl cyclo-tertiary amines using imine reductase, key components in natural products and pharmaceutical synthesis, is a green approach. Traditional methods faced challenges with enzyme activity and selectivity. This study focused on enhancing Mesorhizobium imine reductase (MesIRED) for better N-benzyl cyclo-tertiary amine production. Through alanine scanning and consensus mutation, 12 single-site MesIRED mutants were identified from 23 candidates, showing improved conversion of N-benzylpyrrolidine and N-benzylpiperidine. Notably, mutants from I177, V212, I213, and A241 significantly boosted conversions. The best-performing mutant for N-benzylpyrrolidine, MesIRED^V212A/I213V (M1), increased conversion from 23.7% to 74.3%. For N-benzylpiperidine, MesIRED^{V212A/I177A/A241I} (M2) enhanced conversion from 22.8% to 66.8%. Tunnel analysis revealed M1 and M2 have more efficient tunnels for larger product movement compared to wild-type MesIRED. Using recombinant E. coli coexpressing MesIRED and glucose dehydrogenase (GDH), high conversions were achieved: 75.1% for N-benzylpyrrolidine (M1) and 88.8% for N-benzylpiperidine (M2). A preparative experiment resulted in 86.2% conversion and 60.2% yield for N-benzylpiperidine. This research offers an efficient method for engineering IRED, significantly improving conversion and selectivity for N-benzyl cyclo-tertiary amines, aiding drug synthesis and providing insights into rational design of other enzymes. Full article

Polyamines participate in the processes of cell growth and development. The degradation branch of their metabolism involves amine oxidases. The oxidation of spermine, spermidine and putrescine releases hydrogen peroxide and the corresponding aminoaldehyde. Polyamine-derived aminoaldehydes have been found to be cytotoxic, and they represent the subject of this review. 3-aminopropanal disrupts the lysosomal membrane and triggers apoptosis or necrosis in the damaged cells. It is implicated in the pathogenesis of cerebral ischemia. Furthermore, 3-aminopropanal yields acrolein through the elimination of ammonia. This reactive aldehyde is also generated by the decomposition of aminoaldehydes produced in the reaction of serum amine oxidase with spermidine or spermine. In addition, acrolein is a common environmental pollutant. It causes covalent modifications of proteins, including carbonylation, the production of Michael-type adducts and cross-linking, and it has been associated with inflammation-related diseases. APAL and acrolein are detoxified by aldehyde dehydrogenases and other mechanisms. High-performance liquid chromatography, immunochemistry and mass spectrometry have been largely used to analyze the presence of polyamine-derived aminoaldehydes and protein modifications elicited by their effect. However, the main and still open challenge is to find clues for discovering clear linkages between aldehyde-induced modifications of specific proteins and the development of various diseases. Full article

Introduction: Biogenic amines play important roles throughout cellular metabolism. This study explores a role of biogenic amines in glioblastoma pathogenesis. Here, we characterize the plasma levels of biogenic amines in glioblastoma patients undergoing standard-of-care treatment. Methods: We examined 138 plasma samples from 36 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma at multiple stages of treatment. Untargeted gas chromatography–time of flight mass spectrometry (GC-TOF MS) was used to measure metabolite levels. Machine learning approaches were then used to develop a predictive tool based on these datasets. Results: Surgery was associated with increased levels of 12 metabolites and decreased levels of 11 metabolites. Chemoradiation was associated with increased levels of three metabolites and decreased levels of three other metabolites. Ensemble learning models, specifically random forest (RF) and AdaBoost (AB), accurately classified treatment phases with high accuracy (RF: 0.81 ± 0.04, AB: 0.78 ± 0.05). The metabolites sorbitol and N-methylisoleucine were identified as important predictive features and confirmed via SHAP. Conclusion: To our knowledge, this is the first study to describe plasma biogenic amine signatures throughout the treatment of patients with glioblastoma. A larger study is needed to confirm these results with hopes of developing a diagnostic algorithm. Full article

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (V_max) increased for variant Phe72Thr at the cost of an increased value for the Michaelis–Menten constant (K_m) for histamine. The increased K_m value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class. Full article

A glucose biosensor was layer-by-layer assembled on a modified glassy carbon electrode (GCE) from a nanocomposite of NAD(P)⁺-dependent glucose dehydrogenase, aminated polyethylene glycol (mPEG), carboxylic acid-functionalized multi-wall carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymers. The electrochemical electrode was denoted as NF/IL/GDH/mPEG-fMWCNTs/GCE. The composite polymer membranes were characterized by cyclic voltammetry, ultraviolet-visible spectrophotometry, electrochemical impedance spectroscopy, scanning electron microscopy, and transmission electron microscopy. The cyclic voltammogram of the modified electrode had a pair of well-defined quasi-reversible redox peaks with a formal potential of −61 mV (vs. Ag/AgCl) at a scan rate of 0.05 V s⁻¹. The heterogeneous electron transfer constant (k_s) of GDH on the composite functional polymer-modified GCE was 6.5 s⁻¹. The biosensor could sensitively recognize and detect glucose linearly from 0.8 to 100 µM with a detection limit down to 0.46 μM (S/N = 3) and a sensitivity of 29.1 nA μM⁻¹. The apparent Michaelis–Menten constant (${\mathrm{K}}_{\mathrm{m}}^{\mathrm{app}}$) of the modified electrode was 0.21 mM. The constructed electrochemical sensor was compared with the high-performance liquid chromatography method for the determination of glucose in commercially available glucose injections. The results demonstrated that the sensor was highly accurate and could be used for the rapid and quantitative determination of glucose concentration. Full article

D-serine plays an essential role in the field of medicine and cosmetics. Diaminopimelate dehydrogenase (DAPDH) is a kind of oxidoreductase that can reduce keto acid into the corresponding D-amino acid. Because of its high stereoselectivity and lack of by-product production, DAPDH has become the preferred enzyme for the efficient one-step synthesis of D-amino acids. However, the types of DAPDH with a reductive amination function reported so far are limited. Although the DAPDH from Symbiobacterium thermophilum (StDAPDH) demonstrates reductive amination activity toward a series of macromolecular keto acids, activity toward hydroxypyruvate (HPPA) for D-serine synthesis has not been reported. In this study, we investigated the activity of the available StDAPDH/H227V toward HPPA by measuring the desired product D-serine. After homologous structure modeling and docking analysis concerning the substrate-binding pocket, four residues, D92, D122, M152, and N253, in the active pocket were predicted for catalyzing HPPA. Through single-point saturation mutation and iterative mutation, a mutant D92E/D122W/M152S was obtained with an 8.64-fold increase in enzyme activity, exhibiting a specific activity of 0.19 U/mg and k_cat value of 3.96 s⁻¹ toward HPPA. Using molecular dynamics simulation, it was speculated that the increase in enzyme activity might be related to the change in substrate pocket size and the enhancement of the interactions between the substrate and key residues. Full article

Despite its limited exploration, Nymphaea mexicana Zucc. can be beneficial if pharmacology, isolation, and biological evaluation are given attention. It is an aquatic species that belongs to the family Nymphaeaceae. The thrust area of the work was the extraction, isolation, and biological evaluation of different extracts of the N. mexicana Zucc. plant. The primary goal of this research was to assess the antimicrobial, antioxidant, and anticancer activities of the extracts and to isolate the target naringenin compound. Comparative FT IR analysis of different extracts of this plant revealed the presence of functional groups of plant secondary metabolites, including polyphenols, flavonoids, terpenoids, esters, amines, glycosides, alkanes, alkaloids, fatty acids, and alcohols. Moderate free radical scavenging potential has been achieved for the various extracts via reducing power and DPPH assays. While cytotoxic activity was evaluated by colorimetric and lactate dehydrogenase cell viability tests on potent cancer cell lines. Lung adenocarcinoma epithelial cells (A-549), and breast cells (MC-7) were treated with MeOH extract. The antimicrobial activity against bacterial strains was evaluated using Gram-positive and -negative cultures, where maximum and minimum inhibition zones were recorded for different strains, including 1.6–25.6 μg/mL for Streptococcus aureus, using the agar well diffusion method. In addition, the anti-inflammatory activity of different extracts of N. mexicana Zucc. was evaluated in a nitrite radical scavenging assay with high concentrations of secondary metabolites, which are important against human pathogens and other diseases. Full article

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme. Full article

D-amino acids are valuable building blocks for the synthesis of biologically active compounds and pharmaceuticals. The asymmetric synthesis of chiral amino acids from prochiral ketones using stereoselective enzymes is a well-known but far from exhausted approach for large-scale production. Herein, we investigated a pyridoxal-5′-phosphate-dependent D-amino acid transaminase from Haliscomenobacter hydrossis as a potential biocatalyst for the enzymatic asymmetric synthesis of optically pure aliphatic and aromatic D-amino acids. We studied the catalytic efficiency and stereoselectivity of transaminase from H. hydrossis in the amination of aliphatic and aromatic α-keto acids, using D-glutamate as a source of the amino group. We constructed a one-pot three-enzyme system, which included transaminase and two auxiliary enzymes, hydroxyglutarate dehydrogenase, and glucose dehydrogenase, to produce D-amino acids with a product yield of 95–99% and an enantiomeric excess of more than 99%. We estimated the stability of the transaminase and the cofactor leakage under reaction conditions. It was found that a high concentration of α-keto acids as well as a low reaction temperature (30 °C) can reduce the cofactor leakage under reaction conditions. The obtained results demonstrated the efficiency of transaminase from H. hydrossis in the asymmetric synthesis of enantiomerically pure D-amino acids. Full article

(R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine, a key chiral intermediate of selective tetrodotoxin-sensitive blockers, was efficiently synthesized by a bienzyme cascade system formed by with R-ω-transaminase (ATA117) and an alcohol dehydrogenase (ADH) co-expression system. Herein, we report that the use of ATA117 as the biocatalyst for the amination of 3,5-bistrifluoromethylacetophenone led to the highest efficiency in product performance (enantiomeric excess > 99.9%). Moreover, to further improve the product yield, ADH was introduced into the reaction system to promote an equilibrium shift. Additionally, bienzyme cascade system was constructed by five different expression systems, including two tandem expression recombinant plasmids (pETDuet-ATA117-ADH and pACYCDuet-ATA117-ADH) and three co-expressed dual-plasmids (pETDuet-ATA117/pET28a-ADH, pACYCDuet-ATA117/pET28a-ADH, and pACYCDuet-ATA117/pETDuet-ADH), utilizing recombinant engineered bacteria. Subsequent studies revealed that as compared with ATA117 single enzyme, the substrate handling capacity of BL21(DE3)/pETDuet-ATA117-ADH (0.25 g wet weight) developed for bienzyme cascade system was increased by 1.50 folds under the condition of 40 °C, 180 rpm, 0.1 M pH9 Tris-HCl for 24 h. To the best of our knowledge, ours is the first report demonstrating the production of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine using a bienzyme cascade system, thus providing valuable insights into the biosynthesis of chiral amines. Full article

Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a K_m value of 4.92 mM and a k_cat/K_m value of 24.49 s⁻¹ mM⁻¹. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L⁻¹) L-Tle was achieved with a 96.1% yield and 2.39 g L⁻¹ h⁻¹ productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production. Full article