Search Results (48)

In this study, we investigated the effects of air packaging, vacuum packaging and modified atmosphere packaging (CO₂/N₂: 80/20) on the purine metabolism and enzyme activities of refrigerated large yellow croakers. The results showed that modified atmosphere packaging significantly inhibited microbial growth, delayed adenosine triphosphate degradation and maintained higher IMP content (1.93 μmol/g on day 21) compared to the air packaging group (2.82 μmol/g on day 12). The total purine content increased with storage time, with hypoxanthine content increasing significantly and occupying most of the total content, which was the key factor for the elevation of purine, followed by adenine content showing a significant decreasing trend. Hypoxanthine accumulation was significantly suppressed in the modified atmosphere packaging group (2.31 μmol/g on day 18), which was much lower than that in the air packaging group (5.64 μmol/g), whereas xanthine and guanine did not show significant differences among the groups. The key enzymes xanthine oxidase and purine nucleoside phosphorylase were much less active in modified atmosphere packaging, effectively delaying the cascade reaction of inosine monophosphate → hypoxanthine → xanthine. The study confirmed that modified atmosphere packaging intervenes in purine metabolism through enzyme activity regulation, providing a theoretical basis for the preservation of low purine aquatic products. Full article

The large yellow croaker, or Larimichthys crocea, is highly prized for its golden color and nutritional content. The purpose of this study was to investigate the differences in body composition, mucus biochemical indices and body color in five strains of large yellow croakers (body weight: 347.01 ± 5.86 g). To conduct genetic diversity analyses of the populations, a total of 50 tailfin samples were randomly chosen from the following populations of large yellow croakers: wild (LYC1), Dai-qu population (LYC2), Yongdai 1 (LYC3), Min-yuedong population (LYC4), and Fufa 1 (LYC5). The findings demonstrated that the LYC3 group’s pigment contents, crude protein, crude lipid, and chromatic values were comparable to those of the LYC1 group (p > 0.05). There was no significant difference between the LYC1 and LYC5 groups’ mucus superoxide dismutase (SOD) and catalase (CAT) activities (p > 0.05). The alkaline phosphatases (ALP), acid phosphatases (ACP), and lysozyme (LYS) activities of the mucus in the LYC1 group were not significantly different from the LYC3 group (p > 0.05). The back skin mRNA expressions of tyrosinase (tyr), tyrosinase-related protein 1 (tyrp1), dopachrome tautomerase (dct), microphtalmia-associated transcription factor (mitf), and melanocortin 1 receptor (mc1r) were significantly up-regulated in the LYC2 and LYC4 groups compared to the LYC1, LYC3, and LYC5 groups (p < 0.05). Forkhead box d3 (foxd3), paired box 3 (pax3), purine nucleoside phosphorylase 4a (pnp4a), aristaless-like homeobox 4a (alx4a), cAMP dependent protein kinase (pka), anaplastic lymphoma kinase (alk), leukocyte receptor tyrosine kinase (ltk), and colony stimulating factor (fms) were among the mRNA expressions of the abdominal skin in the LYC1, LYC3, and LYC5 groups significantly higher than those in the LYC2 and LYC4 groups (p < 0.05). In conclusion, the LYC3 group’s crude protein, crude lipid, carotenoid, and lutein contents were most similar to those of the large yellow croaker found in the wild. Furthermore, the molecular mechanism underlying the variations in body color among the various strains of large yellow croakers was supplied for additional research. Full article

A series of new 3′-deoxyribosides of substituted benzimidazoles was obtained by the chemo-enzymatic method using genetically engineered E. coli purine nucleoside phosphorylase (PNP). In the case of asymmetrically substituted benzimidazole derivatives, a mixture of N1- and N3-regioisomers was formed (confirmed by NMR). The antiviral activity of the obtained compounds against herpes simplex virus 1 of reference strain L2 and a strain deeply resistant to acyclovir in Vero E6 cell culture was studied. 4,6-Difluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC₅₀ = 250.92 µM, SI = 12.00) and 4,5,6-trifluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC₅₀ = 249.96 µM, SI = 16.00) showed significant selective activity against both viral models in comparison to ribavirin (IC₅₀ = 511.88 µM, SI > 8.00). Full article

The increasing resistance of Trichomonas vaginalis to the only approved chemical family of drugs for treatment, the 5-nitroimidazoles, has prompted the exploration of new therapeutic agents against this prevalent non-viral sexually transmitted infection. Natural products have emerged as a significant source of novel treatments for trichomoniasis. The aim of this study was to evaluate the anti-T. vaginalis activity of citral (3,7-dimethyl-2,6-octadienal), the main constituent of the essential oil of Cymbopogon species, commonly known as lemongrass. Our findings indicate that citral exhibits a minimum inhibitory concentration (MIC) of 100 μM, effectively inhibiting the growth of T. vaginalis trophozoites within 12 h of exposure, and a 50% inhibitory concentration (IC₅₀) of approximately 40 μM after 24 h. Furthermore, the evaluation of nitric oxide (NO) levels suggests that citral possesses antioxidant properties. Molecular docking studies reveal a weak interaction with three parasite proteins: thioredoxin reductase (TvTrxR), purine nucleoside phosphorylase (TvPNP), and methionine gamma lyase (TvMGL). The present study highlights the potential of citral as a candidate for the development of no-nitroimidazole drugs, offering new avenues for trichomoniasis treatment and underscoring the importance of further investigation into citral’s mechanism of action. Full article

Beer, the most popular alcoholic beverage, poses health risks for individuals with gout and hyperuricemia due to its high purine content. Herein, we identified a novel purine nucleoside phosphorylase (AbPNP) from the edible mushroom Agaricus bisporus and heterologously expressed it in Pichia pastoris. The recombinant AbPNP exhibited optimal activity at 60 °C and pH 7.0, retaining >80% activity at pH 6.0–9.0 and >85% activity after 3 h at ≤60 °C. Kinetic analysis revealed high catalytic efficiency (kcat/Km = 2.02 × 10⁶ s⁻¹⋅M⁻¹) toward inosine, with strong resistance to metal ions except for Co²⁺ and Cu²⁺. The application of AbPNP (1.0–5.0 U/mL) during wort saccharification reduced purine nucleosides by 33.54% (from 151.53 to 100.65 mg/L) while increasing yeast utilization of free purine bases. The resulting beer showed improved fermentation performance (alcohol content increased by 3.6%) without compromising flavor profiles. This study provides the food-grade enzymatic strategy for low-purine beer production, leveraging the GRAS status of both A. bisporus and P. pastoris. Full article

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N²-etheno-2-aminopurine (1,N²-ε2APu, I) and N²,3-etheno-2-aminopurine (N²,3-ε2APu, II). In the present paper, we investigate their interaction with two key enzymes of purine metabolism, purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO), using diffraction of X-rays on protein crystals, isothermal titration calorimetry, and fluorescence spectroscopy. Crystals were obtained and structures were solved for WT PNP and D204N-PNP mutant in a complex with N²,3-ε2APu (II). In the case of WT PNP—1,N²-ε2APu (I) complex, the electron density corresponding to the ligand could not be identified in the active site. Small electron density bobbles may indicate that the ligand binds to the active site of a small number of molecules. On the basis of spectroscopic studies in solution, we found that, in contrast to PNP, 1,N²-ε2APu (I) is the ligand with better affinity to XO. Enzymatic oxidation of (I) leads to a marked increase in fluorescence near 400 nm. Hence, we have developed a new method to determine XO activity in biological material, particularly suitable for milk analysis. Full article

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 °C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. Full article

The enzymatic synthesis of nucleoside derivatives is an important alternative to multi-step chemical methods traditionally used for this purpose. Despite several undeniable advantages of the enzymatic approach, there are a number of factors limiting its application, such as the limited substrate specificity of enzymes, the need to work at fairly low concentrations, and the physicochemical properties of substrates—for example, low solubility. This research conducted by our group is dedicated to the advantages and limitations of using purine nucleoside phosphorylases (PNPs), the main enzymes for the metabolic reutilization of purines, in the synthesis of modified nucleoside analogues. In our work, the substrate specificity of PNP from various bacterial sources (mesophilic and thermophilic) was studied, and the effect of substrate, increased temperature, and the presence of organic solvents on the conversion rate was investigated. Full article

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of “non-typical” minor products of the reaction. In addition to “typical” N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2′-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2′-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this “upside down” arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products. Full article

1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/R^ACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(β-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased. Full article

Chemo-enzymatic syntheses of strongly fluorescent nucleoside analogs, potentially applicable in analytical biochemistry and cell biology are reviewed. The syntheses and properties of fluorescent ribofuranosides of several purine, 8-azapurine, and etheno-purine derivatives, obtained using various types of purine nucleoside phosphorylase (PNP) as catalysts, as well as α-ribose-1-phosphate (r1P) as a second substrate, are described. In several instances, the ribosylation sites are different to the canonical purine N9. Some of the obtained ribosides show fluorescence yields close to 100%. Possible applications of the new analogs include assays of PNP, nucleoside hydrolases, and other enzyme activities both in vitro and within living cells using fluorescence microscopy. Full article

Many enzymes are composed of several identical subunits, which are arranged in a regular fashion and usually comply with some definite symmetry. This symmetry may be approximate or exact and may or may not coincide with the symmetry of crystallographic packing. Purine nucleoside phosphorylases (PNP) are a class of oligomeric enzymes that show an interesting interplay between their internal symmetry and the symmetry of their crystal packings. There are two main classes of this enzyme: trimeric PNPs, or “low-molecular-mass” proteins, which are found mostly in eukaryotic organisms, and hexameric PNPs, or “high-molecular-mass” proteins, which are found mostly in prokaryotic organisms. Interestingly, these two enzyme classes share only 20–30% sequence identity, but the overall fold of the single monomer is similar, yet this monomeric building block results in a different quaternary structure. To investigate this interplay of symmetry in this class of enzymes, a comprehensive database of all PNPs is constructed, containing their local symmetries and interface information. Full article

Both inosine and guanosine are precursors of uric acid that may cause the diseases of hyperuricemia and gout in humans. Here, a promising bacterial strain for efficiently biodegrading both inosine and guanosine was successfully isolated from a healthy human intestine and identified as Bacillus paranthracis YD01 with 16S rRNA analysis. An initial amount of 49.6 mg·L⁻¹ of inosine or 49.9 mg·L⁻¹ of guanosine was completely removed by YD01 within 12 h, which showed that YD01 had a strong ability to biodegrade inosine and guanosine. Furthermore, the initial amount of 49.2 mg·L⁻¹ of inosine or 49.5 mg·L⁻¹ of guanosine was totally catalyzed by the intracellular crude enzymes of YD01 within 6 h, and the initial inosine amount of 49.6 mg·L⁻¹ or guanosine of 49.7 mg·L⁻¹ was biodegraded by the extracellular crude enzymes of YD01 within 9 h. Illumina Hiseq sequencing and database gene annotation were used to elucidate the genomic characteristics of B. paranthracis YD01. Purine nucleoside phosphorylase, encoded by gene 1785, gene 3933, and gene 4403, was found in the KEEG database, which played a crucial role in the biodegradation of inosine and guanosine. The results of this study provide valuable insights into the mechanisms for biodegrading inosine and guanosine using B. paranthracis YD01. Full article

To explore the anti-hyperuricemia components in sunflower (Helianthus annuus L.) calathide extract (SCE), we identified abietic acid (AA) via liquid chromatography–mass spectrometry and found an excellent inhibitor of xanthine oxidase (IC₅₀ = 10.60 µM, Ki = 193.65 nM) without cytotoxicity. Based on the transcriptomics analysis of the human embryonic kidney 293T cell model established using 1 mM uric acid, we evaluated that AA showed opposite modulation of purine metabolism to the UA group and markedly suppressed the intensity of purine nucleoside phosphorylase, ribose phosphate pyrophosphokinase 2, and ribose 5-phosphate isomerase A. Molecular docking also reveals the inhibition of purine nucleoside phosphorylase and ribose phosphate pyrophosphokinase 1. The SCE exhibits similar regulation of these genes, so we conclude that AA was a promising component in SCE against hyperuricemia. This present study provided a novel cell model for screening anti-hyperuricemia natural drugs in vitro and illustrated that AA, a natural diterpenoid, is a potential inhibitor of purine biosynthesis or metabolism. Full article

Ganpu vine tea is a new type of health care citrus fruit tea made from citrus shell, Pu-er tea, and vine tea baked as raw materials. In this study, the in vitro uric acid synthase inhibition system and hyperuric acid cell model were constructed to appraise the uric acid lowering efficacy of Ganpu vine tea, traditional Ganpu tea, and vine tea. Results showed that in the uric acid synthase inhibition system, the aqueous extract can inhibite the puric metabolically related enzymes, such as adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XOD). The ability of the aqueous extract to inhibit the above enzyme was as follows: vine tea > Ganpu vine tea > Ganpu tea; all teas had a strong effect on XOD inhibition. The hyperuric acid cell model test showed that the aqueous extract inhibited uric acid production through accumulating inosine and hypoxanthine and hindering xanthine synthesis. The uric acid reductive ability was as follows: Vine tea > Ganpu vine tea > Ganpu tea. The inhibition of enzymes related to uric acid synthesis and the inhibition of uric acid production were significantly enhanced through adding vine tea to Ganpu tea. It also shows that flavonoids are the main factor driving this ability because they are the main active ingredients in these botanical drinks. Full article