Overexpression, Purification, and Biochemical Characterization of the vanC2 d-Ala-d-Ser Ligase from Enterococcus casseliflavus SSK and Its Inhibition by an Oxadiazole Derivative Sneha B. Paymal, Sagar S. Barale, Shirishkumar V. Supanekar, Kailas D. Sonawane, Kiran D. Pawar ACS Omega, 2025 The bacterial cell wall and enzymes involved in peptidoglycan biosynthesis are prime targets for the discovery of novel antibacterial agents. Among these enzymes, d-alanine-d-alanine ligases (Ddl) are particularly significant due to their utilization of specific substrates (d-amino acids) essential for bacterial viability. Isozymes of Ddl that utilize alternative substrates such as d-lactate or d-serine are found in vancomycin-resistant Gram-positive bacteria, initially identified in Enterococcus species, and now represent a growing concern in clinical settings. In this study, we isolated and identified vancomycin-resistant Enterococcus casseliflavus (E. casseliflavus) strain SSK and used it for amplification, cloning, and purification of the vanC2 type of d-alanine-d-serine ligase (EcfDdls). Investigations of substrate specificity and enzyme kinetics provided insights into the enzyme’s mechanistic action. Evaluation of the inhibitory potential of the previously virtually screened oxadiazole derivative 1-[(5-methyl-1,2-oxazol-3-yl)methyl]-4-{[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]methyl}piperazine (CID 45805715) was carried out using an inorganic phosphate detection assay, which demonstrated complete enzymatic inhibition of purified EcfDdls. When tested, CID 45805715 significantly inhibited activity of Ddl, with an IC50 of 76.7 μM, compared to 313 μM for the reference compound DCS. Moreover, this compound also exhibited antimicrobial activity against vancomycin-resistant E. casseliflavus strain SSK. Thus, these findings provide valuable insights into the activity and inhibition of vanC2 EcfDdls, offering a promising avenue for addressing vancomycin resistance in enterococci, particularly in nosocomial infections affecting immunocompromised patients.
Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques Subodh A. Kamble, Sagar S. Barale, Ali Abdulmawjood Mohammed, Sneha B. Paymal, Nitin M. Naik, Kailas D. Sonawane Amino Acids, 2024 Alzheimer’s disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aβ) peptides. The extracellular deposition of Aβ peptides in human AD brain causes neuronal death. Therefore, it has been found that Aβ peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD.
Purification and characterization of antibacterial surfactin isoforms produced by Bacillus velezensis SK Sagar S. Barale, Savaliram G. Ghane, Kailas D. Sonawane AMB Express, 2022 Bacillus velezensis SK having broad-spectrum antimicrobial activity has been isolated from soil. The efficient extraction of antimicrobial compounds produced in various mediums has been done using Diaion HP-20 resin. Further, characterization of an antimicrobial compound by TLC, FTIR, in-situ bioautography analysis revealed the presence of cyclic lipopeptides, which is then purified by the combination of silica gel, size exclusion, dual gradient, and RP-HPLC chromatography techniques. Growth kinetic studies showed that Bacillus velezensis SK produces a mixture of lipopeptides (1.33 gL−1). The lipopeptide exhibits good pH (2–10) and temperature stability up to 80 °C. LC–ESI–MS analysis of partially purified lipopeptide identified variant of surfactin, further analysis of purified chromatographic fractions revealed the occurrence of most abundant C15-surfactin homologues (m/z 1036.72 Da). The isolated surfactin exhibits good antimicrobial activity (1600 AU/ml) against drug-resistant food-born B. cereus and human pathogen Staphylococcus aureus. Hence, identified strain B. velezensis SK and its potent antibacterial surfactin lipopeptide could be used in various food and biomedical applications.
Production, isolation and characterization of exotoxin produced by Bacillus subtilis, Bacillus megaterium and Proteus vulgaris and its significance in food poisoning International Journal of Pharmaceutical Sciences Review and Research, 2015
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Polymyxin-B as a novel inhibitor of amyloid beta aggregation: Computational insights and experimental validation AA Mohammed, SS Barale, PS Dhotare, KD Sonawane Journal of Molecular Structure, 143738 , 2025 2025 Citations: 6
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