Search Results (10)

In this research, we sought to methodically examine the protective effects of Gastrodia elata extract (GEE) on liver damage induced by D-galactose (D-gal) in mice and clarify the underlying mechanisms. The chemical composition of GEE was characterized using Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS), while network pharmacology analysis was employed to predict potential molecular targets and signaling pathways. A mouse model of liver injury was established through daily intraperitoneal injection of D-gal over a 42-day period, during which the hepatoprotective efficacy of GEE was evaluated. Biochemical, histopathological, and molecular analyses were subsequently performed. UPLC-MS/MS identified ingredients such as amino acids, aromatic compounds, fatty acids, and terpenoids in GEE. A network pharmacology analysis enabled the identification of 272 common targets linked to GEE and liver damage, demonstrating notable enrichment within the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. In vivo experiments demonstrated that GEE effectively alleviated D-gal-induced body weight loss and elevated liver index values, alleviated hepatic histological damage, and reduced serum levels of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Alkaline Phosphatase (ALP). Furthermore, GEE enhanced the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), decreased malondialdehyde (MDA) levels, and downregulated the mRNA expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β), and Tumor Necrosis Factor-alpha (TNF-α). Western blot analysis confirmed that GEE activated the PI3K/Akt pathway, as evidenced by increased ratios of phosphorylated Phosphatidylinositol 3-kinase/Phosphatidylinositol 3-kinase (p-PI3K/PI3K) and phosphorylated AKT/Protein Kinase B (p-AKT/AKT); restored the B-cell lymphoma 2-associated X protein/B-cell lymphoma-2 (Bax/Bcl-2) balance; and reduced cyclin-dependent kinase inhibitor 1 (p21) expression. The results suggest that GEE protects against D-gal-induced liver damage by reducing oxidative stress, inhibiting inflammatory responses, and modulating apoptosis through the activation of the PI3K/Akt signaling pathway, providing support for its potential use in hepatoprotection. Full article

Formed by L-phenylalanine (L-phe) ammonia under the action of aromatic amino acid aminotransferases (AAATs), volatile benzenoids (VBs) and volatile phenylpropanoids (VPs) are essential aromatic components in oolong tea (Camellia sinensis). However, the key VB/VP components responsible for the aromatic quality of oolong tea need to be revealed, and the formation mechanism of VBs/VPs based on AAAT branches during the post-harvest process of oolong tea remains unclear. Therefore, in this study, raw oolong tea and manufacturing samples were used as the test materials, and targeted metabolomics combined with transcriptome analysis was also conducted. The results showed that thirteen types of VBs/VPs were identified, including nine types of VPs and four types of VBs. Based on the OAV calculation, in raw oolong tea, 2-hydroxy benzoic acid methyl ester and phenylethyl alcohol were identified as key components of the aromatic quality of oolong tea. As for the results from the selection of related genes, firstly, a total of sixteen candidate CsAAAT genes were selected and divided into two sub-families (CsAAAT1 and CsAAAT2); then, six key CsAAAT genes closely related to VB/VP formation were screened. The upregulation of the expression level of CsAAAT2-type genes may respond to light stress during solar-withering as well as the mechanical force of turnover. This study can help to understand the formation mechanism of aromatic compounds during oolong tea processing and provide a theoretical reference for future research on the formation of naturally floral and fruity aromas in oolong tea. Full article

The chemotherapy drug doxorubicin (DOX) is an anthracycline with over 30% incidence of liver injury in breast cancer patients, yet the mechanism of its hepatotoxicity remains unclear. To identify potential biomarkers for anthracycline-induced hepatotoxicity (AIH), we generated clinically-relevant mouse and rat models administered low-dose, long-term DOX. These models exhibited significant liver damage but no decline in cardiac function. Through untargeted metabolic profiling of the liver, we identified 27 differential metabolites in a mouse model and 28 in a rat model. We then constructed a metabolite-metabolite network for each animal model and computationally identified several potential metabolic markers, with particular emphasis on aromatic amino acids, including phenylalanine, tyrosine, and tryptophan. We further performed targeted metabolomics analysis on DOX-treated 4T1 breast cancer mice for external validation. We found significant (p < 0.001) reductions in hepatic levels of phenylalanine and tyrosine (but not tryptophan) following DOX treatment, which were strongly correlated with serum aminotransferases (ALT and AST) levels. In summary, the results of our study present compelling evidence supporting the use of phenylalanine and tyrosine as metabolic signatures of AIH. Full article

Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an amino donor to a keto substrate. ATs are promising biocatalysts that are replacing traditional chemical routes for the production of chiral amines. In this study, an in silico-screening of a metagenomic library isolated from the Curonian Lagoon identified 11 full-length fold type IV aminotransferases that were successfully expressed and used for substrate profiling. Three of them (AT-872, AT-1132, and AT-4421) were active toward (R)-methylbenzylamine. Purified proteins showed activity with L- and D-amino acids and various aromatic compounds such as (R)-1-aminotetraline. AT-872 and AT-1132 exhibited thermostability and retained about 55% and 80% of their activities, respectively, even after 24 h of incubation at 50 °C. Active site modeling revealed that AT-872 and AT-4421 have an unusual active site environment similar to the AT of Haliscomenobacter hydrossis, while AT-1132 appeared to be structurally related to the AT from thermophilic archaea Geoglobus acetivorans. Thus, we have identified and characterized PLP fold type IV ATs that were active toward both amino acids and a variety of (R)-amines. Full article

To secure a sustainable food supply for the rapidly growing global population, great efforts towards a plant-based diet are underway. However, the use of plant proteins comes with several challenges, such as improvement or removal of undesired flavours, and generation of desired texture properties. Fermentation holds large potential to alter these properties, but compared to dairy fermentations, our knowledge on strain properties in different plant-based substrates is still limited. Here, we explored different lactic acid bacteria for their ability to grow, produce flavour compounds, or remove off-flavour compounds from different plant proteins. For this, 151 LAB strains from dairy and non-dairy origins were cultured in plant protein plus coconut oil emulsions supplemented with glucose. Pea, chickpea, mung, fava, and soybean proteins were used in the study and bacterial strains for screening included the genera Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc. Efficient, high throughput, screening on plant proteins was developed and strains were assessed for their ability to (i) acidify and decrease the pH; (ii) express key enzymes involved in the formation of amino acid derived flavours, which included PepN (aminopeptidase N), PepXP (X-prolyl dipeptidyl peptidase), EstA (esterase), BcAT (branched chain aminotransferase), CBL (cystathione beta lyase), and ArAT (aromatic aminotransferase); and (iii) improve the overall aroma profile by generating dairy/cheesy notes and decreasing off flavours. Suitable screening conditions were determined, and highlighted the importance that a sufficient heat treatment must be applied to samples containing plant proteins, prior to fermentation, as an outgrowth of spore forming Bacillus cereus was observed if the material was only pasteurised. Enzyme activities for strains measured in rich broth vs. a buffered protein solution showed little-to-no correlation, which illustrated the importance of screening conditions to obtain predictive enzyme measurements. Aroma formation analysis allowed to identify strains that were able to increase key aromas such as diacetyl, acetoin, 2- and 3-methyl butanol, and 2,3-pentanedione, as well as decrease the off-flavours hexanal, pentanal, and nonanal. Our findings illustrate the importance of strain specific differences in the assessed functionalities and how a methodical approach to screening LAB can be applied to select suitable microorganisms that show promise in fermentation of plant proteins when applied in non-dairy cheese applications. Full article

Photorespiration is a metabolic process that removes toxic 2-phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. It is essential for plant growth under ambient air, and it can play an important role under stress conditions that reduce CO₂ entry into the leaf thus enhancing photorespiration. The aim of the study was to determine the impact of photorespiration on Arabidopsis thaliana leaf amino acid metabolism under low atmospheric CO₂ concentrations. To achieve this, wild-type plants and photorespiratory glycolate oxidase (gox) mutants were given either short-term (4 h) or long-term (1 to 8 d) low atmospheric CO₂ concentration treatments and leaf amino acid levels were measured and analyzed. Low CO₂ treatments rapidly decreased net CO₂ assimilation rate and triggered a broad reconfiguration of soluble amino acids. The most significant changes involved photorespiratory Gly and Ser, aromatic and branched-chain amino acids as well as Ala, Asp, Asn, Arg, GABA and homoSer. While the Gly/Ser ratio increased in all Arabidopsis lines between air and low CO₂ conditions, low CO₂ conditions led to a higher increase in both Gly and Ser contents in gox1 and gox2.2 mutants when compared to wild-type and gox2.1 plants. Results are discussed with respect to potential limiting enzymatic steps with a special emphasis on photorespiratory aminotransferase activities and the complexity of photorespiration. Full article

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors’ binding allows for explanation of the enzyme’s adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding. Full article

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan catabolism, acts as an antagonist for both the α7 nicotinic acetylcholine receptor and glycine coagonist sites of the N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Elevation of brain kynurenic acid levels reduces the release of neurotransmitters such as dopamine and glutamate, and kynurenic acid is considered to be involved in psychiatric disorders such as schizophrenia and depression. Thus, the control of kynurenine pathway, especially kynurenic acid production, in the brain is an important target for the improvement of brain function or the effective treatment of brain disorders. Astrocytes uptake kynurenine, the immediate precursor of kynurenic acid, via large neutral amino acid transporters, and metabolize kynurenine to kynurenic acid by kynurenine aminotransferases. The former transport both branched-chain and aromatic amino acids, and the latter have substrate specificity for amino acids and their metabolites. Recent studies have suggested the possibility that amino acids may suppress kynurenic acid production via the blockade of kynurenine transport or via kynurenic acid synthesis reactions. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with elevated kynurenic acid levels. Full article

The aminotransferase from Bacillus circulans (BtrR), which is involved in the biosynthesis of butirosin, catalyzes the pyridoxal phosphate (PLP)-dependent transamination reaction to convert valienone to β-valienamine (a new β-glycosidase inhibitor for the treatment of lysosomal storage diseases) with an optical purity enantiomeric excess value. To explore the stereoselective mechanism of valienamine generated by BtrR, multiple molecular dynamics (MD) simulations were performed for the BtrR/PLP/valienamine and BtrR/PLP/β-valienamine complexes. The theoretical results showed that β-valienamine could make BtrR more stable and dense than valienamine. β-valienamine could increase the hydrogen bond probability and decrease the binding free energy between coenzyme PLP and BtrR by regulating the protein structure of BtrR, which was conducive to the catalytic reaction. β-valienamine maintained the formation of cation-p interactions between basic and aromatic amino acids in BtrR, thus enhancing its stability and catalytic activity. In addition, CAVER 3.0 analysis revealed that β-valienamine could make the tunnel of BtrR wider and straight, which was propitious to the removal of products from BtrR. Steered MD simulation results showed that valienamine interacted with more residues in the tunnel during dissociation compared with β-valienamine, resulting in the need for a stronger force to be acquired from BtrR. Taken together, BtrR was more inclined to catalyze the substrates to form β-valienamine, either from the point of view of the catalytic reaction or product removal. Full article

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases. Full article