Search Results (13)

To explore the differences in protein quality among classic medicinal entomopathogenic fungi and to evaluate their metabolic adaptability, we analyzed the amino acid composition and proteomic characteristics of Cordyceps sinensis (CS), Cordyceps militaris (CM), and Cordyceps cicadae (CC). Quantitative analysis showed CM contained the highest crude protein and lysine, methionine, threonine, and valine. CS adapted to high-altitude hypoxia and exhibited lower protein but elevated leucine, isoleucine, and histidine contents, which may contribute to membrane stabilization and oxidative stress resistance. CC displayed higher non-essential amino acids such as arginine, proline, and tyrosine, reflecting active nitrogen metabolism. Four-dimensional data-independent acquisition (4D-DIA) proteomics identified 495 differentially expressed proteins (DEPs). Compared with CS, CM and CC displayed upregulated glutamate oxaloacetate transaminases 2 (GOT2), glutamate dehydrogenase (GDH), and argininosuccinate synthase 1 (ASS1) coordinately regulate nitrogen flux through the alanine-aspartate-glutamate metabolic network and urea cycle, supporting metabolic intermediate replenishment for energy metabolism. The upregulation of branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA) and acyl-CoA dehydrogenase short/branched chain (ACADSB) in CM and CC facilitated the integration of branched-chain amino acid catabolism with the TCA cycle, explaining species-specific differences in protein content. This study presents the first application of 4D-DIA proteomics to compare CS, CM, and CC, providing insights into quality divergence mechanisms in medicinal fungi. Full article

This study investigated the potential of dietary gamma-aminobutyric acid (GABA) inclusion to mitigate acute temperature stress impacting the physiological resilience of juvenile olive flounder (Paralichthys olivaceus). A total of 360 juvenile fish, with an average initial weight of 12.97 ± 0.1 g (mean ± SEM), were randomly assigned in triplicate to 18 tanks (20 fish per tank) and reared at 19.5 °C for 8 weeks, with bi-monthly collection of growth performance data. The fish were fed one of six experimental diets: control (GABA74), 174 ppm of GABA (GABA174), 275 ppm of GABA (GABA275), 396 ppm of GABA (GABA396), 476 ppm of GABA (GABA476), and 516 ppm of GABA (GABA516). At the end of the trial, one group of fish was subjected to lethal temperature stress (31 °C) for 48 h, while another was exposed to acute temperature stress (29 °C) for 6 h. Growth performance remained relatively stable across all inclusion levels (p > 0.05), with the final body weight (FBW) ranging from 48.2 ± 0.3 g (GABA174) to 50.3 ± 0.6 g (GABA516) and the feed conversion ratio (FCR) varying between 2.06 ± 0.07 (GABA396) and 2.35 ± 0.07 (control). There were no significant differences in average whole-body composition across all dietary treatments, with moisture content ranging from 74.8 to 75.0%, crude protein from 17.8 to 18.2%, crude lipid from 2.89 to 3.15%, and crude ash from 3.62 to 3.80%. Similarly, there were no significant differences in cumulative survival rates during lethal temperature exposure between the GABA-supplemented groups and the control group, with an average of 28.5 ± 4.6%. Additionally, GABA inclusion did not significantly alter plasma-free amino acid profiles, antioxidant enzyme activities, or immune functions (p > 0.05). However, temperature significantly reduced the levels of superoxide dismutase (SOD) from 3.34 ± 0.17 to 2.29 ± 0.36 µg/mL and increased the levels of glutamate oxaloacetate transaminase (GOT) from 17.1 ± 0.8 to 46.3 ± 6.2 U/L, glutamate pyruvate transaminase (GPT) from 14.4 ± 0.6 to 30.2 ± 2.1 U/L, glucose (GLU) from 13.3 ± 0.5 to 68.7 ± 7.7 mg/dL, total protein (TP) from 2.94 ± 0.00 to 3.21 ± 0.1 g/dL, and cortisol from 5001 ± 147 to 6395 ± 194 ng/mL. Furthermore, no significant changes were observed in the expression of key stress-related genes, including heat shock proteins (hsp60, hsp70, and hsp90) and the warm water acclimation-related gene wap65. This study establishes the safety of GABA as a dietary inclusion for olive flounder and highlights its potential to enhance stress resilience in aquaculture. However, the effectiveness of GABA-based interventions could depend on critical factors such as dosage, stress duration, and species-specific responses. Our findings highlight the need for further research to optimize GABA inclusion strategies, particularly with consideration for long-term physiological impacts. Full article

The first monomeric pyridoxal-5′-phosphate (PLP)-dependent transaminase from a marine, aromatic-compound-degrading, sulfate-reducing bacterium Desulfobacula toluolica Tol2, has been studied using structural, kinetic, and spectral methods. The monomeric organization of the transaminase was confirmed by both gel filtration and crystallography. The PLP-dependent transaminase is of the fold type IV and deaminates D-alanine and (R)-phenylethylamine in half-reactions. The enzyme shows high stereoselectivity; no deamination of L-amino acids and (S)-phenylethylamine is detected. Structural analysis and subsequent mutagenesis led to the conclusion that the monomeric architecture of the enzyme is the only one possible and sufficient for stereoselectivity and PLP binding, but not for the overall double-substrate transamination reaction and the stability of the holo form with the reduced cofactor—pyridoxamine-5′-phosphate. These results extend the structural university of the PLP fold type IV enzymes and demonstrate the need for deeper analysis of the sequence–structure–function relationships in the transaminases. Full article

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5′-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure–function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine—a reversible inhibitor and analogue of (R)-1-phenylethylamine—a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds. Full article

Pyridoxal-5′-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism. Full article

Staphylococcus aureus is regarded as a threatening bacterial pathogen causing invasive pneumonia in healthcare settings and in the community. The continuous emergence of multidrug resistant strains is narrowing the treatment options for these infections. The development of an effective S. aureus vaccine is, therefore, a global priority. We have previously developed a vaccine candidate, 132 ΔmurI Δdat, which is auxotrophic for D-glutamate, and protects against sepsis caused by S. aureus. In the present study, we explored the potential of this vaccine candidate to prevent staphylococcal pneumonia, by using an acute lung infection model in BALB/c mice. Intranasal inoculation of the vaccine strain yielded transitory colonization of the lung tissue, stimulated production of relevant serum IgG and secretory IgA antibodies in the lung and distal vaginal mucosa and conferred cross-protection to acute pneumonia caused by clinically important S. aureus strains. Although these findings are promising, additional research is needed to minimize dose-dependent toxicity for safer intranasal immunization with this vaccine candidate. 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

A specific mosquito enzyme, 3-hydroxykynurenine transaminase (HKT), is involved in the processing of toxic metabolic intermediates of the tryptophan metabolic pathway. The HKT enzymatic product, xanthurenic acid, is required for Plasmodium spp. development in the mosquito vectors. Therefore, an inhibitor of HKT may not only be a mosquitocide but also a malaria-transmission blocker. In this work, we present a study investigating the evolution of HKT, which is a lineage-specific duplication of an alanine glyoxylate aminotransferases (AGT) in mosquitoes. Synteny analyses, together with the phylogenetic history of the AGT family, suggests that HKT and the mosquito AGTs are paralogous that were formed via a duplication event in their common ancestor. Furthermore, 41 amino acid sites with significant evidence of positive selection were identified, which could be responsible for biochemical and functional evolution and the stability of conformational stabilization. To get a deeper understanding of the evolution of ligands’ capacity and the ligand-binding mechanism of HKT, the sequence and the 3D homology model of the common ancestor of HKT and AGT in mosquitoes, ancestral mosquito AGT (AncMosqAGT), were inferred and built. The homology model along with 3-hydroxykynurenine, kynurenine, and alanine were used in docking experiments to predict the binding capacity and ligand-binding mode of the new substrates related to toxic metabolites detoxification. Our study provides evidence for the dramatic biochemical evolution of the key detoxifying enzyme and provides potential sites that could hinder the detoxification function, which may be used in mosquito larvicide and design. Full article

d-Aspartate oxidase (DDO) is a peroxisomal flavoenzyme that catalyzes the oxidative deamination of acidic d-amino acids. In the yeast Cryptococcus humicola strain UJ1, the enzyme ChDDO is essential for d-Asp utilization and is expressed only in the presence of d-Asp. Pyruvate carboxylase (Pyc) catalyzes the conversion of pyruvate to oxaloacetate and is involved in the import and activation of certain peroxisomal flavoenzymes in yeasts. In this study, we analyzed the role of Pyc in the expression of ChDDO gene in C. humicola strain UJ1. PYC gene disruption (∆Chpyc1) in strain UJ1 resulted in growth retardation on glucose and NH₄Cl medium. The growth was restored by supplying oxaloacetate from l-Asp or α-ketoglutarate by a transaminase. On the other hand, the supply of oxaloacetate from d-Asp by ChDDO was not able to prevent growth retardation because of a significant decrease in ChDDO gene expression at the transcriptional level. The addition of pyruvate significantly decreased ChDDO gene transcription in the ∆Chpyc1 strain but increased the same in the wild-type strain, even though the intracellular pyruvate content was similar in both strains. These results suggest that ChDDO gene expression might be regulated by pyruvate metabolism, as well as by the presence of d-Asp. Full article

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes. Full article

In this study, we investigated the effect of dietary supplementation with acetate and L-tryptophan-conjugated bypass amino acid (ACT) during late pregnancy on the production performance of cows pre- and postpartum and their offspring. Eight multiparous Holstein cows (calving date ±15 d, 2nd parity; n = 4) were supplied with diets without ACT supplementation (Control) or with 15 g/day ACT supplementation (ACT). The results showed that ACT improved the feed intake (FI) in dry cows. No differences in blood hematological parameters were found between the two groups of prepartum cows. The serum glutamic-oxaloacetic transaminase activity increased and the triglyceride concentration decreased in the ACT-treated group compared to the control group. In the postpartum cows, milk compositions were not affected by ACT supplementation. Saturated fatty acid (SFA) content in the colostrum was significantly lower in the ACT-treated group than in the control group. Serum glucose (GLC) level was significantly higher in the ACT-treated group than in the control group. Monocyte and GLC levels were lower in calves of groups where their dams had received ACT. Overall, we found higher FI in the dry cows, lower colostrum SFA levels, and heavier calf birth weight (5.5 kg) when the dams were supplemented with ACT, suggesting a positive nutrient compensation by ACT supplementation to dry cows. Full article

Concerns regarding environmental exposures and the impacts of pharmaceuticals on non-target aquatic organisms continue to increase. The antiepileptic drug carbamazepine (CBZ) is often detected as an aquatic contaminant and can disrupt various behaviors of fishes. However, there are few reports which investigate the mechanism of CBZ action in fish. The aim of the current study was to evaluate the effects of CBZ on embryonic development (i.e., hatching rate, heart rate, and body length) and early spontaneous movement. Moreover, we sought to investigate potential mechanisms by focusing on the gamma-aminobutyric acid (GABA) neurotransmitter system in zebrafish 6 days after of exposure. The results show that CBZ exposure did not cause significant effects on embryo development (hatching rate, heart rate, nor body length) at the test concentrations. However, the early spontaneous movement of embryos was inhibited following 10 μg/L CBZ exposure at 28–29 h post-fertilization (hpf). In addition, acetylcholinesterase (AChE) activity and GABA concentrations were increased with exposure, whereas glutamate (Glu) concentrations were decreased in larval zebrafish. Gene expression analysis revealed that GABA and glutamate metabolic pathways in zebrafish larvae were altered following exposure to CBZ. GABA transaminase (abat) and glutamic acid decarboxylase (gad1b) decreased to 100 µg/L, and glutamate receptor, ionotropic, N-methyl D-aspartate 1b (grin1b) as well as the glutamate receptor, ionotropic, α-amino-3hydroxy-5methylisoxazole-4propionic 2b (gria2b) were down-regulated with exposure to 1 µg/L CBZ. Our study suggests that CBZ, which can act as an agonist of the GABA_A receptor in humans, can also induce alterations in the GABAergic system in fish. Overall, this study improves understanding of the neurotoxicity and behavioral toxicity of zebrafish exposed to CBZ and generates data to be used to understand mechanisms of action that may underlie antiepileptic drug exposures. Full article

Aim: Patients on parenteral nutrition (PN) are prone to inflammation. This may aggravate an existing proinflammatory state and become a critical factor in the development of liver dysfunction (LD). Intravenous fish oil may attenuate this inflammatory state, but data on its use in adults are scarce. The aim of this study was to investigate the effects of adding a pure fish oil intravenous lipid emulsion (ILE) into short- and long-term PN in patients either at risk of, or with existing, inflammation. Methods: A retrospective analysis of 61 patients (32 female, 29 male, mean age 51.5 ± 12.6 years) who received all-in-one PN, including amino acids, glucose, and lipids supplemented with pure fish oil ILE, was performed. Pure fish oil ILE (Omegaven®, Fresenius Kabi, Bad Homburg, Germany) was used along with the standard ILE to reach a fish oil dose of 0.4–0.5 g fish oil/kg/d. Diagnoses were chronic intestinal failure (CIF, n = 20), Crohn’s disease (CD, n = 22), and ulcerative colitis (UC, n = 19). The observation period was 12 months for CIF and 21 days for UC and CD. Results: A reduction in inflammation was noticeable in all patients and became statistically significant in CD (hsCRP p < 0.0001, ESR p = 0.0034, procalcitonin p = 0.0014, Il-6 p = 0.001) and UC groups (hsCRP and ESR p < 0.0001, Il-6 p = 0.0001, TNF-α p = 0.0113). In the CIF group, the total bilirubin concentration (p = 0.2157) and aspartate transaminase SGOT (p = 0.1785) did not vary over time. Conclusions: PN with pure fish oil ILE reduces some inflammatory parameters in IBD and maintains liver function parameters in CIF patients. Fish oil might become a valuable ingredient in both short- and long-term PN in patients at risk of liver dysfunction. Full article