Small-angle X-ray scattering studies of the open and closed conformations of aspartate aminotransferase

Summary Aspartate aminotransferase was investigated by X-ray small-angle scattering. A small difference was found between the open (active) and the closed (liganded) conformation of the enzyme. The results were compared with X-ray crystallography data.

---

Untersuchungen zur Röntgenkleinwinkelstreuung der offenen und geschlossenen Konformation von Aspartat-Aminotransferase

Zusammenfassung Aspartat-Aminotransferase wurde mittels Röntgenkleinwinkelstreuung untersucht. Ein kleiner Unterschied zwischen der offenen (aktiven) und der geschlossenen (ligandierten) Konformation wurde gefunden. Die Ergebnisse wurden mit Röntgenkristallstrukturdaten verglichen.

---

Abbreviations AspAT aspartate aminotransferase     

Astragalus membranaceus ultrafine powder alleviates hyperuricemia by regulating the gut microbiome and reversing bile acid and adrenal hormone biosynthesis dysregulation

Ethnopharmacological relevanceMetabolic syndrome is closely related to the intestinal microbiota and disturbances in the host metabolome. Hyperuricemia (HUA), a manifestation of metabolic syndrome, can induce various cardiovascular diseases and gout, seriously affecting a patient’s quality of life. Astragalus membranaceus has a long history as a commonly used traditional Chinese medicine to treat kidney disease in China and East Asia.Materials and methodsWe compared the therapeutic effect of benzbromarone and two different doses Astragalus membranaceus ultrafine powder (AMUP) in rats with HUA. Ultra-performance liquid chromatography-mass spectrometer was used to analyze the AMUP metabolism in the plasma, urine, and feces. Further, 16S ribosome RNA sequencing and feces metabolomic were performed to capture the variation of the gut microbiota and metabolites changes before and after drug administration.ResultsAMUP had a notable impact on reducing blood uric acid levels while protecting the liver and kidney. Drug metabolism analysis demonstrated that effective constituent flavonoids are distributed in the blood, whereas saponins remain in the intestine. Gut microbiota analysis showed that low-dose AMUP ameliorated HUA-induced gut dysbiosis by reducing the abundance of harmful bacteria and increasing that of some beneficial bacteria with anti-inflammatory properties, such as Clostridia, Lachnospiraceae, and Muribaculaceae. In addition, HUA-induced changes in metabolite contents in bile acid and adrenal hormone biosynthesis pathways were restored after treatment with AMUP.ConclusionLow-dose AMUP exerts remarkable therapeutic effects on HUA by regulating the gut microbiome and mediating gut metabolism pathways associated with uric acid excretion.     

Conformational dependence of the intrinsic acidity of the aspartic acid residue sidechain in N-acetyl- -aspartic acid-N′-methylamide

The sidechain conformational potential energy hypersurfaces (PEHS) for the γ_L, β_L, α_L, and α_D backbone conformations of N-acetyl- -aspartate-N′-methylamide were generated. Of the 81 possible conformers initially expected for the aspartate residue, only seven were found after geometric optimizations at the B3LYP/6-31G(d) level of theory. No stable conformers could be located in the δ_L, _L, γ_D, δ_D, and _D backbone conformations. The ‘adiabatic’ deprotonation energies for the endo and exo forms of N-acetyl- -aspartic acid-N′-methylamide were calculated by comparing their optimized relative energies against those found for the seven stable conformers of N-acetyl- -aspartate-N′-methylamide. Sidechain conformational PEHSs were also generated for the estimation of ‘vertical’ deprotonation energies for both endo and exo forms of N-acetyl- -aspartic acid-N′-methylamide. All backbone–sidechain (N–H⁻O–C) and backbone–backbone (N–HO=C) hydrogen bond interactions were analyzed. A total of two backbone–backbone and four backbone–sidechain interactions were found for N-acetyl- -aspartate-N′-methylamide. The deprotonated sidechain of N-acetyl- -aspartate-N′-methylamide may allow the aspartyl residue to form strong hydrogen bond interactions (since it is negatively charged) which may be significant in such processes as protein–ligand recognition and ligand binding. As a primary example, the molecular geometry of the aspartyl residue may be important in peptide folding, such as that in the RGD tripeptide.     

高效液相色谱法测定丝氨酸:乙醛酸转氨酶的活性

报道了一种用反相高效液相色谱法(HPLC)测定丝氨酸:乙醛酸转氨酶(SGAT,EC2.6.1.45)活性变化的有效方法。将酶初提液与含有L-丝氨酸和乙醛酸的反应体系在30 ℃下反应1 h,反应终止后进行2,4-二硝基氟苯衍生化,通过HPLC测定酶反应前后甘氨酸的衍生化产物量的变化来分析酶的活性。通过流动相组成和浓度梯度的优化,既节省了流动相的用量,也避免了气泡的产生。研究结果表明,该测定方法的准确度和灵敏度比分光光度法和纸色谱分析法高,且简便易行;对于其他转氨酶活性的测定也有一定的参考价值。     

Simultaneous liquid chromatographic determination of metals and organic compounds in pharmaceutical and food-supplement formulations using evaporative light scattering detection

A novel method for the non-derivatization liquid chromatographic determination of metals (potassium, aluminium, calcium and magnesium) and organic compounds (ascorbate and aspartate) was developed and validated based on evaporative light scattering detection (ELSD). Separation of calcium, magnesium and aluminium was achieved by the cation exchange column Dionex CS-14 and an aqueous TFA mobile phase according to the following time program: 0-6 min TFA 0.96 mL L⁻¹, 6-7 min linear gradient from TFA 0.96-6.4 mL L⁻¹. Separation of potassium, magnesium and aspartate was achieved by the lipophilic C₁₈ Waters Spherisorb column and isocratic aqueous 0.2 mL L⁻¹ TFA mobile phase. Separation of sodium, magnesium, ascorbate and citrate was also achieved by the C₁₈ analytical column, according to the following elution program: 0-2.5 min aqueous nonafluoropentanoic acid (NFPA) 0.5 mL L⁻¹; 2.5-3.5 min linear gradient from 0.5 mL L⁻¹ NFPA to 1.0 mL L⁻¹ TFA. In all cases, evaporation temperature was 70 °C, pressure of the nebulizing gas (nitrogen) 3.5 bar, gain 11 and the flow rate 1.0 mL min⁻¹. Resolution among calcium and magnesium was 1.8, while for all other separations was ≥3.2. Double logarithmic calibration curves were obtained within various ranges from 3-24 to 34-132 μg mL⁻¹, and with good correlation (r > 0.996). Asymmetry factor ranged from 0.9 to 1.9 and limit of detection from 1.3 (magnesium) to 17 μg mL⁻¹ (ascorbate).The developed method was applied for the assay of potassium, magnesium, calcium, aluminium, aspartate and ascorbate in pharmaceuticals and food-supplements. The accuracy of the method was evaluated using spiked samples (%recovery 95-105%, %R.S.D. < 2) and the absence of constant or proportional errors was confirmed by dilution experiments.     

Preliminary Results on the Crystal and Molecular Structure of Native and Mutant Aspartate Carbamoyltransferase at Neutral pH

The cell dimensions are a=b=131.2, c=198.5 Å, and α= β =90°, γ = 120° for both the wild and the mutant types of aspartate transcarbamoylase (ATCase) crystallized at pH=7.0. The space group is R32. Although the mutant and wild forms of ATCase show large differences in enzyme activity and chemical reactivity, yet they are isomorphous in structure. One asymmetric unit of ATCase contains a catalytic subunit, molecular weight 34,000 daltons, and a regulatory subunit, molecular weight 17,000. There are 310 amino acids in the catalytic subunit and 162 amino acids plus zinc in the regulatory subunit. The whole ATCase is a hexamer and consists of six molecules of catalytic and regulatory subunits. The primary structure study of this enzyme shows that the mutant form has a single substitution of aspartic acid in place of glycine at position 128 in the amino acid sequence of the catalytic chains. In most part of the sequence, the regulatory and catalytic chains of mutant and wild forms of ATCase are in similar conformations. (2Fo-Fc) syntheses displayed with computer graphics using the FRODO program, were applied to the reflection data from both mutant and wild types. A stereoview of certain regions displayed on PS300 graphics shows significant differences between the wild and mutant types of ATCase.     

The latest research progress on the prevention of storage pests by natural products: Species,mechanisms, and sources of inspiration

The quality of grains is influenced by storage pests, which are not only spoilers of stored grain, but also vectors of human and animal diseases. Chemical pesticides play an essential role in the cultivation and storage of cereals, however, due to the low degradability and residual toxicity of synthetic pesticides on the environment and non-target organisms, as well as the increasing resistance of target organisms to them, consideration should be given to the development of alternative pest control agents. Compounds isolated from natural sources have emerged as preferred targets for the development of novel insecticidal agents because of their eco-friendliness, safety, and effectiveness. In this review, we primarily focus on the natural product (NPs) control of storage pests. The effective monomer components of NPs and their anti-insect mechanisms were discussed, and natural sources of inspiration and models for insect repellents are described. This review aimed to provide guidelines for the exploitation and utilization of green and efficient natural insecticides.     

The stir-frying can reduce hepatorenal toxicity of Fructus Tribuli by inactivating β-glucosidase and inhibiting the conversion of furostanol saponins to spirostanol saponins

The purpose of this study was to illustrate the mechanism of “enzyme inactivation and toxicity reduction” of Fructus Tribuli (FT) after being heating processed. Ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) was used to quantitatively analyze the contents of four steroidal saponins in crude Fructus Tribuli (CFT) and stir-fried Fructus Tribuli (SFT) under different storage times at room temperature. The enzyme activity of β-D-glucosidase in CFT and SFT were determined and calculated by ultraviolet–visible spectrometry (UV-VIS spectrometry). In addition, the enzyme hydrolysates of FOT and tribuluside A were qualitatively analyzed by ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). The hepatorenal toxicity of spirostanol saponins in FT were further confirmed by in vivo and in vitro experiment. This study confirmed that “enzyme inactivation and toxicity reduction” was one of the reasons why the stir-frying can reduce hepatorenal toxicity of FT, and further enriched the exploration on the mechanism of processing toxicity reduction.     

The ethnobotanical,phytochemistry and pharmacological activities of Psidium guajava L.

Psidium guajava L., commonly known as guava is an important tropical food plant with diverse medicinal values. In traditional medicine, it is used in the treatment of various diseases such as diarrhoea, diabetes, rheumatism, ulcers, malaria, cough, and bacterial infections. The aim of this review is to provide up-to-date information on the ethnomedicinal uses, bioactive compounds, and pharmacological activities of P. guajava with greater emphasis on its therapeutic potentials. The bioactive constituents extracted from P. guajava include phytochemicals (gallic acid, casuariin, catechin, chlorogenic acid, rutin, vanillic acid, quercetin, syringic acid, kaempferol, apigenin, cinnamic acid, luteolin, quercetin-3-O-α-L-arabinopyranoside, morin, ellagic acid, guaijaverin, pedunculoside, asiastic acid, ursolic acid, oleanolic acid, methyl gallate and epicatechin) and essential oils (limonene, trans-caryophyllene, α-humulene, γ-muurolene, selinene, caryophyllene oxide, bisabolol, isocaryophyllene, δ-cadinene, α-copaene, α-cedrene, β-eudesmol, α-pinene, β-pinene, β-myrcene, linalool, α-terpineol and eucalyptol). In vitro and in vivo studies demonstrated that P. guajava possesses pharmacological activities such as antidiabetic, antidiarrhoeal, hepatoprotective, anticancer, antioxidant, anti-inflammatory, antiestrogenic, and antibacterial activities which support its traditional uses. The exhibited pharmacological activities reported may be attributed to the numerous bioactive compounds present in different parts of P. guajava. Based on the beneficial effects of P. guajava as well as its bioactive constituents, it can be exploited in the development of pharmaceutical products and functional foods. However, there is a need for comprehensive studies in clinical trials to establish the safe doses and efficacy of P. guajava for the treatment of several diseases.     

Spectrophotometric enzymatic cycling method using -glutamate dehydrogenase and -phenylglycine aminotransferase for determination of -glutamate in foods

This report describes a new spectrophotometric method capable of determining low levels of -glutamate. The assay is based on substrate cycling between -glutamate dehydrogenase (GlDH) and the novel enzyme -phenylglycine aminotransferase ( -PhgAT). In this system, GlDH converts -glutamate to 2-oxoglutarate with concomitant reduction of NAD⁺ to NADH. The 2-oxoglutarate is recycled to -glutamate in a transamination reaction catalyzed by -PhgAT using -4-hydroxyphenylglycine as an amino donor, which is converted to 4-hydroxybenzoylformate. Both NADH and 4-hydroxybenzoylformate strongly absorb UV light at 340 nm (_340nm=6.22×10³ and 8.90×10³ l mol⁻¹ cm⁻¹, respectively). The signal amplification effect of the cycling reactions is thus further enhanced by the combined absorption of the two accumulating reaction products. The standard calibration curve for -glutamate was linear from 0.2 to 20 μM, with a detection limit of 0.14 μM. Food samples can be significantly diluted before subjected to the assay, thus reducing the effects of interfering substances. Because of the unique substrate specificity of -PhgAT, -glutamate could be selectively determined in the presence of other common amino acids at relatively high concentrations. The assay was satisfactorily applied to measure -glutamate in various kinds of food products. The procedure is simple, rapid, accurate, and should be easily automated.