Leucine zipper as a bridge for transaminase self-assembly: A fusion enzyme for efficient chiral conversion of d-phenylglycine.

Amino acid transferase is a family of enzymes used to catalyze and separate chiral amino acids. However, due to the low efficiency, by-products and reverse reactions occur in cascade reactions. Therefore, in the research, phenylglycine aminotransferase and aspartate aminotransferase were self-assembled in vitro by leucine zipper. The self-assembled enzyme system with d-phenylglycine and α-ketoglutarate as substrates were used for the chiral transformation reaction. By studying the enzyme combination, kinetic reaction stability and catalytic efficiency, it was found that the self-assembled enzyme showed improved stability and better affinity to the substrate than the control and achieved only ee value of 17.86% for the control at the substrate ratio was 1:2. In contrast, the self-assembled enzyme basically catalyzed the complete conversion of d-Phg to l-Phg, with the ee value as 99%. These results demonstrated the feasibility of the leucine zipper and the conversion of d-phenylglycine to the l-type by fusion enzyme.

The Age-AST-D Dimer (AAD) Regression Model Predicts Severe COVID-19 Disease.

AIM: Coronavirus disease (COVID-19) ranges from mild clinical phenotypes to life-threatening conditions like severe acute respiratory syndrome (SARS). It has been suggested that early liver injury in these patients could be a risk factor for poor outcome. We aimed to identify early biochemical predictive factors related to severe disease development with intensive care requirements in patients with COVID-19. METHODS: Data from COVID-19 patients were collected at admission time to our hospital. Differential biochemical factors were identified between seriously ill patients requiring intensive care unit (ICU) admission (ICU patients) versus stable patients without the need for ICU admission (non-ICU patients). Multiple linear regression was applied, then a predictive model of severity called Age-AST-D dimer (AAD) was constructed (n = 166) and validated (n = 170). RESULTS: Derivation cohort: from 166 patients included, there were 27 (16.3%) ICU patients that showed higher levels of liver injury markers (P < 0.01) compared with non-ICU patients: alanine aminotrasnferase (ALT) 225.4 ± 341.2 vs. 41.3 ± 41.1, aspartate aminotransferase (AST) 325.3 ± 382.4 vs. 52.8 ± 47.1, lactic dehydrogenase (LDH) 764.6 ± 401.9 vs. 461.0 ± 185.6, D-dimer (DD) 7765 ± 9109 vs. 1871 ± 4146, and age 58.6 ± 12.7 vs. 49.1 ± 12.8. With these finding, a model called Age-AST-DD (AAD), with a cut-point of <2.75 (sensitivity = 0.797 and specificity = 0.391, c - statistic = 0.74; 95%IC: 0.62-0.86, P < 0.001), to predict the risk of need admission to ICU (OR = 5.8; 95% CI: 2.2-15.4, P = 0.001), was constructed. Validation cohort: in 170 different patients, the AAD model < 2.75 (c - statistic = 0.80 (95% CI: 0.70-0.91, P < 0.001) adequately predicted the risk (OR = 8.8, 95% CI: 3.4-22.6, P < 0.001) to be admitted in the ICU (27 patients, 15.95%). CONCLUSIONS: The elevation of AST (a possible marker of early liver injury) along with DD and age efficiently predict early (at admission time) probability of ICU admission during the clinical course of COVID-19. The AAD model can improve the comprehensive management of COVID-19 patients, and it could be useful as a triage tool to early classify patients with a high risk of developing a severe clinical course of the disease.

Pimpinella anisum Essential Oil Nanoemulsion Toxicity against Tribolium castaneum? Shedding Light on Its Interactions with Aspartate Aminotransferase and Alanine Aminotransferase by Molecular Docking.

The insecticidal activity is the result of a series of complex interactions between toxic substances as ligands and insect's enzymes as targets. Actually, synthetic insecticides used in pest control programs are harmful to the environment and may affect non-target organisms; thus, the use of natural products as pest control agents can be very attractive. In the present work, the toxic effect of aniseed (Pimpinella anisum L.) essential oil (EO) and its nanoemulsion (NE) against the red flour beetle Tribolium castaneum, has been evaluated. To assess the EO mode of action, the impact of sub-lethal concentrations of aniseed EO and NE was evaluated on enzymatic and macromolecular parameters of the beetles, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, total lipids and glucose. Finally, a molecular docking study was conducted to predict the mode of action of the major EO and NE components namely E-anethole, Limonene, alpha-himalachalene, trans-Verbenol and Linalool at binding site of the enzymes AST and ALT. Herein, the binding location of the main compounds in both proteins are discussed suggesting the possible interactions between the considered enzymes and ligands. The obtained results open new horizons to understand the evolution and response of insect-plant compounds interactions and their effect predicted at the molecular levels and side effects of both animal and human.

Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases.

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways.

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α-carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.

Heterologous gene expression and characterization of recombinant aspartate aminotransferase from Geobacillus thermopakistaniensis.

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 µmol min-1 mg-1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.

Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis.

Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.

Conformational transitions induced by γ-amino butyrate binding in GabR, a bacterial transcriptional regulator.

GabR from Bacillus subtilis is a transcriptional regulator of the MocR subfamily of GntR regulators. The MocR architecture is characterized by the presence of an N-terminal winged-Helix-Turn-Helix domain and a C-terminal domain folded as the pyridoxal 5'-phosphate (PLP) dependent aspartate aminotransferase (AAT). The two domains are linked by a peptide bridge. GabR activates transcription of genes involved in γ-amino butyrate (GABA) degradation upon binding of PLP and GABA. This work is aimed at contributing to the understanding of the molecular mechanism underlying the GabR transcription activation upon GABA binding. To this purpose, the structure of the entire GabR dimer with GABA external aldimine (holo-GABA) has been reconstructed using available crystallographic data. The structure of the apo (without any ligand) and holo (with PLP) GabR forms have been derived from the holo-GABA. An extensive 1 µs comparative molecular dynamics (MD) has been applied to the three forms. Results showed that the presence of GABA external aldimine stiffens the GabR, stabilizes the AAT domain in the closed form and couples the AAT and HTH domains dynamics. Apo and holo GabR appear more flexible especially at the level of the HTH and linker portions and small AAT subdomain.

Aspulvinone O, a natural inhibitor of GOT1 suppresses pancreatic ductal adenocarcinoma cells growth by interfering glutamine metabolism.

BACKGROUND: Distinctive from their normal counterparts, cancer cells exhibit unique metabolic dependencies on glutamine to fuel anabolic processes. Specifically, pancreatic ductal adenocarcinoma (PDAC) cells rely on an unconventional metabolic pathway catalyzed by aspartate transaminase 1 (GOT1) to rewire glutamine metabolism and support nicotinamide adenine dinucleotide phosphate (NADPH) production. Thus, the important role of GOT1 in energy metabolism and Reactive Oxygen Species (ROS) balance demonstrates that targeting GOT1 may serve as an important therapeutic target in PDAC. METHODS: To assay the binding affinity between Aspulvinone O (AO) and GOT1 proteins, the virtual docking, microscale thermophoresis (MST), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) methods were employed. GOT1 was silenced in several PDAC cell lines. The level of OCR and ECR were assayed by seahorse. To evaluate the in vivo anti-tumor efficacy of AO, the xenograft model was built in CB17/scid mouse. RESULTS: Screening of an in-house natural compound library identified the AO as a novel inhibitor of GOT1 and repressed glutamine metabolism, which sensitizes PDAC cells to oxidative stress and suppresses cell proliferation. Virtual docking analysis suggested that AO could bind to the active site of GOT1 and form obvious hydrophobic interaction with Trp141 together with hydrogen bonds with Thr110 and Ser256. Further in vitro validation, including MST, CETSA and DARTS, further demonstrated the specific combining capacity of AO. We also show that the selective inhibition of GOT1 by AO significantly reduces proliferation of PDAC in vitro and in vivo. CONCLUSIONS: Taken together, our findings identify AO as a potent bioactive inhibitor of GOT1 and a novel anti-tumour agent for PDAC therapy.

Crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe.

L-aspartate aminotransferase is a pyridoxal 5'-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open-closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5'-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5'-phosphate-dependent enzymes.

Persistent Isolated Elevation of Aspartate Aminotransferase in an Asymptomatic Female Patient: a Case Report and Review of Current Literature.

BACKGROUND: Persistent isolated elevation of aspartate aminotransferase (AST) is a rare observation and might lead to unnecessary laboratory testing and invasive procedures, if the possibility of macro-AST is not considered. METHODS: We report the case of a healthy 28-year-old female patient with persistent isolated elevation of AST. In order to confirm the suspected diagnosis of macro-AST, polyethylene glycol (PEG) precipitation and repeated measurements of enzyme activity after refrigeration at 2 - 8°C were performed. RESULTS: PEG precipitation confirmed the presence of macro-AST, while repeated measurements after refrigeration did not show any relevant decrease in enzyme activity. CONCLUSIONS: Especially in clinically asymptomatic patients, macro-AST must be considered as a cause of persistent isolated elevations in AST activity to avoid costly and potentially harmful medical tests or procedures. PEG precipitation is a feasible and cost-effective way to establish the diagnosis, while repeated measurement of enzyme activity after refrigeration potentially leads to wrong conclusions.

Biodistribution and Systemic Effects in Mice Following Intravenous Administration of Cadmium Telluride Quantum Dot Nanoparticles.

Quantum dots (QDs) are engineered nanoparticles (NPs) of semiconductor structure that possess unique optical and electronic properties and are widely used in biomedical applications; however, their risks are not entirely understood. This study investigated the tissue distribution and toxic effects of cadmium telluride quantum dots (CdTe-QDs) in male BALB/c mice for up to 1 week after single-dose intravenous injections. CdTe-QDs were detected in the blood, lung, heart, liver, spleen, kidney, testis and brain. Most CdTe-QDs accumulated in the liver, followed by the spleen and kidney. At high doses, exposure to CdTe-QDs resulted in mild dehydration, lethargy, ruffled fur, hunched posture, and body weight loss. Histological analysis of the tissues, upon highest dose exposures, revealed hepatic hemorrhage and necrotic areas in the spleen. The sera of mice treated with high doses of CdTe-QDs showed significant increases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels, as well as a reduction in albumin. CdTe-QD exposure also led to a reduced number of platelets and elevated total white blood cell counts, including monocytes and neutrophils, serum amyloid A, and several pro-inflammatory cytokines. These results demonstrated that the liver is the main target of CdTe-QDs and that exposure to CdTe-QDs leads to hepatic and splenic injury, as well as systemic effects, in mice. By contrast, cadmium chloride (CdCl2), at an equivalent concentration of cadmium, appeared to have a different pharmacokinetic pattern from that of CdTe-QDs, having minimal effects on the aforementioned parameters, suggesting that cadmium alone cannot fully explain the toxicity of CdTe-QDs.

Oxidative stress, genotoxicity, biochemical and histopathological modifications induced by epoxiconazole in liver and kidney of Wistar rats.

Epoxiconazole (EPX) is a triazole fungicide commonly used in agriculture and for domestic purposes around the world. The excessive application of this pesticide may result in a variety of adverse effects on non-target organisms, including humans. Since, the liver and kidneys are the target organs of this fungicide, potential hepatotoxic and nephrotoxic effects are of high relevance. Thus, our study aimed to investigate the toxic effects of EPX on the liver and kidney of Wistar rats. The exposure of rats to EPX at these concentrations (8, 24, 40, 56 mg/kg bw representing, respectively, NOEL (no observed effect level), NOEL × 3, NOEL × 5, and NOEL × 7) for 28 days significantly enhances hepatic and renal lipid peroxidation which is accompanied by an increase in the level of protein oxidation. Furthermore, the results of the present study clearly indicated that EPX administration induces an increase in the levels of DNA damage in a dose-dependent manner. In addition, the activities of liver and kidney antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) are increased significantly in EPX-treated rats at concentrations of 8, 24, and 40 mg/kg bw. However, with the dose NOEL × 7 (56 mg/kg bw of EPX), the activities of CAT, GPx, and GST are decreased. Indeed, EPX-intoxicated rats revealed a significant reduction in acetylcholinesterase (AChE) activity in both liver and kidney compared with the control group. Also, our results demonstrated that the EPX administration leads to a disruption of the hepatic (aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH)) and renal (uric acid and creatinine) functions. The biochemical perturbations obtained in the present study are corroborated with the histopathological modifications. Since EPX treatment caused severe damage in the overall histo-architecture of liver and kidney tissues, these results suggest that administration of EPX induced a marked deregulation of liver and kidney functions. Graphical abstract.

The protective efficacy of soursop fruit extract against hepatic injury associated with acetaminophen exposure is mediated through antioxidant, anti-inflammatory, and anti-apoptotic activities.

In the current report, we examined the potential beneficial role of soursop fruit extract (SSFE) on liver injury induced by a single paracetamol (APAP) overdose (2000 mg/kg). Thirty-five Wistar albino rats were randomly divided into five groups as follows: control, SSFE, APAP, SSFE+APAP, and silymarin (SIL)+APAP. APAP intoxication was found to elevate alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin levels. Moreover, it increased the levels of malondialdehyde, nitrites, and nitrates and depleted glutathione, superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. APAP intoxication inactivated the nuclear factor erythroid 2-related factor 2 (Nrf2) defense pathway and upregulated the expression of heme oxygenase-1 (HO-1). APAP administration enhanced the activation of nuclear factor-kappa B (NF-κB), the elevation of tumor necrosis factor-alpha and interleukin 1-beta levels, and the upregulation of inducible nitric oxide synthase mRNA expression. In addition, APAP activated the overexpression of Bax protein, increased release of cytochrome c, and the downregulation of Bcl-2 protein. Finally, APAP-induced overexpression of transforming growth factor-beta (TGF-ß) further suggested enhanced liver damage. On the other hand, SSFE pretreatment attenuated these biochemical, molecular, and histopathological alterations in the liver, which might be partially due to the regulation of hepatic Nrf2/HO-1 and downregulation of NF-κB and TGF-ß.

Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1ß aspartate aminotransferase.

Alternative routes for the post-chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4-hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1ß aspartate aminotransferase (1ß AAT), N-succinyl-l,l-diaminopimelate aminotransferase, and branched-chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1ß AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X-ray crystal structure analysis of two PAT competent 1ß AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1ß AAT from R. meliloti. This structural analysis supported by site-directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N-terminal loop of 1ß AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1ß AAT), present only in PAT competent enzymes, could interact with the 4-hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1ß AAT.

Isolated elevation of aspartate aminotransferase (AST) in an asymptomatic patient due to macro-AST.

BACKGROUND: A rare and benign cause of isolated aspartate aminotransferase (AST) increase is due to the presence of macro aspartate aminotransferase (macro-AST). Macro-AST is not usually considered by clinicians, leading to a large number of unnecessary and even invasive tests before diagnosed. Most commonly used laboratory methods for detection of macro-enzymes are precipitation with polyethylene glycol (PEG), ultracentrifugation and gel filtration chromatography (GFC). METHODS: We report a case of an asymptomatic patient with persistent isolated AST elevation. PEG precipitation and an alternative method based on the low stability of this macro-enzyme were performed on the patient serum. RESULTS: Macro-AST was early detected by the laboratory observing a significant decrease in AST concentrations when the serum is stored at 4ºC. CONCLUSIONS: Macro-AST detection based in this method can be very useful as screening, especially in those laboratories where other technologies are not available. Macro-AST must be considered as cause of unexplained isolated AST elevations before prior investigations to avoid the consequent cost and potential harm to the patient. In our case, early detection of macro-AST with this method avoided unnecessary treatment and invasive test such as liver biopsy.

Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods.

The aim of the study was to establish normal ranges for chosen biochemical parameters of haemolymph of snails (Gasropoda: Mollusca), in the light of the use of these animals as experi- mental models in various types of studies. The study was conducted on 100 specimens of Cornu aspersum maxima (CAM) and 100 specimens of Cepaea nemoralis (CN). The haemolymph col- lected from the animals was analysed using colorimetry to assay aspartate transaminase (AST) activity, alanine transaminase (ALT) activity, amylase activity and the concentrations of urea and triglycerides. In the further part of the study, the influence of administering doxycycline with feed on the change of AST and ALT activity in snail haemolymph has been studied. The normal values established for CAM are as follow: AST activity: 26-38 u/l, ALT activity: 0-11 u/l, amylase activity 9-16 u/l, concentration of urea: 3-6 mg/dl, concentration of triglycerides: 16-20 mg/dl. For CN, the following data have been obtained: AST activity: 30-80 u/l, ALT activity: 0-15 u/l, amylase activity 12-15 u/l, concentration of urea: 5-8 mg/dl, concentration of triglycerides: 18-24 mg/dl. It has been shown that doxycycline presents a high workload on the hepatopancreas of snails, which is reflected by a statistically significant (p ⟨ 0.05) increase of AST and ALT activity in the haemolymph of the specimens which obtained doxycycline in feed, as compared to the groups with antibiotic-free feed. The haemolymph activity of both studied parameters increased together with study time and tetracycline administration time.