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Background: Paenibacillus thiaminolyticus, a species of genus Paenibacillus of the family Paenibacillaceae, exists widely in environments and habitats in various plants and worms, and occasionally causes human infections. This work aimed to characterize the function of a novel aminoglycoside O-nucleotidyltransferase resistance gene, designated ant(6)-If, from a P. thiaminolyticus strain PATH554. Methods: Molecular cloning, antimicrobial susceptibility testing, enzyme expression and purification, and kinetic analysis were used to validate the function of the novel gene. Whole-genome sequencing and comparative genomic analysis were performed to investigate the phylogenetic relationship of ANT(6)-If and other aminoglycoside O-nucleotidyltransferases, and the synteny of ant(6)-If related sequences. Results: The recombinant with the cloned ant(6)-If gene (pMD19-ant(6)-If/ DH5α) demonstrated a 128-fold increase of minimum inhibitory concentration level against streptomycin, compared with the control strains (DH5α and pMD19/DH5α). The kinetic parameter kcat/Km of ANT(6)-If for streptomycin was 9.01 × 10³  M−1 ·s−1 . Among the function-characterized resistance genes, ANT(6)- If shared the highest amino acid sequence identity of 75.35% with AadK. The ant(6)-If gene was located within a relatively conserved genomic region in the chromosome. Conclusion: ant(6)-If conferred resistance to streptomycin. The study of a novel resistance gene in an unusual environmental bacterium in this work contributed to elucidating the resistance mechanisms in the microorganisms. [ABSTRACT FROM AUTHOR]

Natural products are an important source of drug development and have good prospects in the treatment of many diseases. However, the efficacy of many natural drugs is achieved through multi-targets, which poses a huge obstacle to their development. In this study, a bienzyme microcapsule system based on α-amylase and α-glucosidase was constructed for the ligand fishing of natural compounds. The microcapsules were formed on the surface of calcium carbonate particles by self-polymerization of dopamine, which physically encapsulated iron oxide and α-glucosidase. Then, α-amylase was immobilized on the surface of magnetic material with glutaraldehyde as crosslinking agent, and calcium carbonate template was removed by EDTA to form a bienzyme system for affinity screening of active components from Toona sinensis. The results showed that two compounds, 1,2,3,4,6-penta-O-galloyl-β- D -glucose and quercitrin were affinity screened from the extracts of Toona sinensis. In vitro activity experiment proved that they were α-amylase and α-glucosidase inhibitors, respectively. Moreover, both of them were mixed type inhibitors that can interact with active centers of corresponding enzymes through multiple hydrogen bonds, van der Waals forces, etc. This study not only proved the effectiveness of the bienzyme system, but also provided a successful example for multi-target screening of natural products. [Display omitted] • Bifunctional microcapsules of α-amylase and α-glucosidase was constructed. • Active components were dual-target affinity screened by microcapsules. • Interactions between targets and active compounds were studied by molecular docking. [ABSTRACT FROM AUTHOR]

In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella , which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH_ 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 μM, and 5.999 ± 1.24 μM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 μg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs. • Successfully cloned the full-length ORF of Eimeria tenella 2-Methylcitrate synthesis (EtPrpC) encoding gene. • EtPrpC has 2-Methylcitrate synthase enzyme activity, and it has weak catalytic activity towards acetyl-CoA. • E. tenella has a 2-Methylcitrate cycle pathway and can serve as a potential target for anti-coccidian drugs. [ABSTRACT FROM AUTHOR]

Ten acridones isolated from Atalantia monophylla were evaluated for effects on Alzheimer’s disease pathogenesis including antioxidant effects, acetylcholinesterase (AChE) inhibition, prevention of beta-amyloid (Aβ) aggregation and neuroprotection. To understand the mechanism, the type of AChE inhibition was investigated in vitro and binding interactions between acridones and AChE or Aβ were explored in silico. Drug-likeness and ADMET parameters were predicted in silico using SwissADME and pKCSM programs, respectively. All acridones showed favorable drug-likeness and possessed multifunctional activities targeting AChE function, Aβ aggregation and oxidation. All acridones inhibited AChE in a mixed-type manner and bound AChE at both catalytic anionic and peripheral anionic sites. In silico analysis showed that acridones interfered with Aβ aggregation by interacting at the central hydrophobic core, C-terminal hydrophobic region, and the key residues 41 and 42. Citrusinine II showed potent multifunctional action with the best ADMET profile and could alleviate neuronal cell damage induced by hydrogen peroxide and Aβ1-42 toxicity.

Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope effects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with different rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly different contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment.

Background: Paenibacillus thiaminolyticus , a species of genus Paenibacillus of the family Paenibacillaceae , exists widely in environments and habitats in various plants and worms, and occasionally causes human infections. This work aimed to characterize the function of a novel aminoglycoside O -nucleotidyltransferase resistance gene, designated ant(6)-If , from a P. thiaminolyticus strain PATH554., Methods: Molecular cloning, antimicrobial susceptibility testing, enzyme expression and purification, and kinetic analysis were used to validate the function of the novel gene. Whole-genome sequencing and comparative genomic analysis were performed to investigate the phylogenetic relationship of ANT(6)-If and other aminoglycoside O -nucleotidyltransferases, and the synteny of ant(6)-If related sequences., Results: The recombinant with the cloned ant(6)-If gene (pMD19- ant(6)-If /DH5α) demonstrated a 128-fold increase of minimum inhibitory concentration level against streptomycin, compared with the control strains (DH5α and pMD19/DH5α). The kinetic parameter k cat /K m of ANT(6)-If for streptomycin was 9.01 × 10 3  M -1 ·s -1 . Among the function-characterized resistance genes, ANT(6)-If shared the highest amino acid sequence identity of 75.35% with AadK. The ant(6)-If gene was located within a relatively conserved genomic region in the chromosome., Conclusion: ant(6)-If conferred resistance to streptomycin. The study of a novel resistance gene in an unusual environmental bacterium in this work contributed to elucidating the resistance mechanisms in the microorganisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lu, Sha, Gao, Shi, Lin, Li, Bao and Feng.)

Abstract: l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k cat and K m values of 685s−1 and 0.27mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg2+), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements. [Copyright &y& Elsevier]

Ten acridones isolated from Atalantia monophylla were evaluated for effects on Alzheimer's disease pathogenesis including antioxidant effects, acetylcholinesterase (AChE) inhibition, prevention of beta-amyloid (Aβ) aggregation and neuroprotection. To understand the mechanism, the type of AChE inhibition was investigated in vitro and binding interactions between acridones and AChE or Aβ were explored in silico. Drug-likeness and ADMET parameters were predicted in silico using SwissADME and pKCSM programs, respectively. All acridones showed favorable drug-likeness and possessed multifunctional activities targeting AChE function, Aβ aggregation and oxidation. All acridones inhibited AChE in a mixed-type manner and bound AChE at both catalytic anionic and peripheral anionic sites. In silico analysis showed that acridones interfered with Aβ aggregation by interacting at the central hydrophobic core, C-terminal hydrophobic region, and the key residues 41 and 42. Citrusinine II showed potent multifunctional action with the best ADMET profile and could alleviate neuronal cell damage induced by hydrogen peroxide and Aβ1-42 toxicity. [ABSTRACT FROM AUTHOR]

Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope effects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with different rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly different contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment. [ABSTRACT FROM AUTHOR]