Biology subjects

KUMAR, P.

Publications and source records attributed to KUMAR, P..

4 recordsLinked to original sources

bioRxiv2026

Structural basis of substrate recognition and conformational gating in the bacteriophage M15 metalloendopeptidase LysPH

The rapid emergence of antimicrobial resistance has led to a surge in multidrug-resistant strains, jeopardising the efficacy of frontline and last-resort antibiotics and thereby aggravating the antimicrobial resistance crisis. Bacteriophage-derived endolysins represent a promising class of next-generation antimicrobials. Here, we report the isolation of a novel bacteriophage, PA_Ganga_001, targeting multidrug-resistant Pseudomonas aeruginosa. Genomic sequencing of this phage identified a previously uncharacterised endolysin LysPH, a zinc-dependent globular endolysin that exhibits antibacterial activity against multiple multidrug-resistant Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Site-directed mutagenesis of the endolysin LysPH suggests that His77, Asp84, His159, Arg41, and Asp156 are essential for catalysis and substrate accommodation. To understand the structural basis of substrate binding and catalysis, we determined the crystal structures of the apo-enzyme (1.8 [A]) and the catalytically attenuated D156A mutant in complex with a synthetic pentapeptide stem (PGX) of peptidoglycan (2.0 [A]). The pentapeptide-bound complex revealed a defined substrate-binding groove, and a pronounced displacement of the Thr42-Ser75 loop was observed. This led to an approximately six-fold volumetric expansion of the catalytic cleft, indicative of a substrate-induced transition to an open, catalytically competent conformation. Notably, a distinctive C-terminal helical element, which diverges sequentially from characterised M15 homologues, is predicted to interact with the NAG-NAM scaffold and may contribute to the positioning of the stem pentapeptide for Zn2+ dependent catalysis. These distinct structural features establish the molecular basis for L-Ala-D-Glu hydrolysis by M15 bacteriophage endolysins and provide a foundational framework for the rational design and engineering of the next generation of antimicrobial enzymes.

biochemistry↗

bioRxiv2025

Unveiling the PET plastics degradation potential of the thermostable EstS1 esterase through integrated biochemical, structural, and morphological analyses

Enzymatic polyethylene terephthalate (PET) plastic degradation is a promising approach to combat the exploding plastic pollution. EstS1, a pH-tolerant, thermostable esterase, has been previously recognized for its degradation potential against phthalate diester plasticizers. The present study elucidates the exceptional potential of this enzyme to degrade crystalline PET plastic and its primary intermediate, bis(2-hydroxyethyl)terephthalate (BHET), into terephthalate. Kinetic analyses revealed that EstS1 degrades 75% of BHET in 1h, liberating mono(2-hydroxyethyl) terephthalate (MHET) and terephthalate as end products. The co-crystal structure of wild-type EstS1 with BHET exhibited the electron density of BHET, MHET, and ethylene glycol, including MHET bound at the active site, in a canonical tetrahedral intermediate conformation. The complex structure of BHET with the Ser154Ala mutant of EstS1 further accommodated two BHET molecules, one interacting directly with the catalytic triad and the oxyanion hole. MD simulation analysis revealed highly stable interactions of BHET at the active site of EstS1. Moreover, SEM imaging displayed significant degradation of the crystalline PET plastic film by EstS1 esterase over a period of 15 days, both under controlled and soil-based fluctuating environmental conditions, highlighting its versatility to varying environmental conditions. XPS analysis discovered the increase in -C-O-, -C-N-, -N-H-, and -N=O- bonds at the surface of EstS1-treated PET film, indicating effective degradation. Consequently, this comprehensive kinetic, structural, and morphology-based analysis of the PET-degrading potential of EstS1 esterase encourages further enzyme engineering studies to exploit the dual potential of EstS1 esterase to degrade both plastic and plasticizers.

biophysics↗

bioRxiv2025

Unveiling the structural insights of PFAS-β-lactoglobulin binding mechanism mediating neuronal toxicity in neonates.

The strong, polar-covalent nature of C-F bonds contributes to the forever nature of per- and polyfluoroalkyl (PFAS) substances. PFAS are toxic to humans. Here, we have examined the ability of the small, globular, milk protein {beta}-lactoglobulin to bind PFAS. The protein transports hydrophobic and amphiphilic compounds, including retinol and fatty acids, for vision and brain development; therefore, underscoring its interactions with PFAS is significant. The crystal structures of {beta}-lactoglobulin complexed with PFOA (Perfluorooctanoic acid) at 2.0 ([A]), PFOS (Perfluorooctanesulfonic acid) at 2.5 ([A]), and PFDA (Perfluorodecanoic acid) at 2.0 ([A]) reveal high affinity of the compounds for the central calyx of {beta}-lactoglobulin, which is the canonical retinol and fatty acid binding site. Analyses of the data indicate significant hydrophobic interactions stabilizing the binding of the PFAS hydrophobic "tails" within the calyx and interactions between Lys60 and Lys69 and PFAS polar head groups. Comparative structural analysis revealed the presence of an open conformation of the EF loop containing the Glu89 latch residue in the complexed structures vis-a-vis the apo-form. Molecular dynamics (MD) simulations revealed high stability of the PFAS binding and attainment of energy minima in all complexes. The average binding energy of PFDA in {beta}-lactoglobulin calyx was -25 kcal/mol, which was higher than PFOS (-21 kcal/mol) and PFOA (-23 kcal/mol) due to increased van der Waals interactions of the longer hydrophobic chain of PFDA with {beta}-lactoglobulin. This work advances a mechanism by which {beta}-lactoglobulin can recruit PFAS and act as a transporter for the "forever" chemical, potentially mediating its neurotoxicity.

biophysics↗

bioRxiv2024

Unveiling Mechanistic and Structural Insights of EstS1 Esterase: A Potent Broad-Spectrum Phthalate Diester Degrading Enzyme

The ubiquitous presence of plastics and plasticizers around the globe has raised an alarming condition. Phthalate diesters are high-priority pollutants that mimic natural hormones and act as endocrine disruptors upon entering living systems. While certain bacterial esterases have been identified for their role in phthalate diester degradation, their structural and mechanistic characteristics remain largely unexplored. A thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus catalyzes the conversion of low molecular weight phthalate diesters to monoesters. This study highlights the unique potential of EstS1 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by employing biophysical and biochemical approaches along with in-depth structural analysis utilizing high-resolution crystal structures in both apo and complex forms, with various substrates, products, and their analogs to elucidate mechanistic details. The catalytic tunnel mediating entry and exit of the substrate and product, respectively, centralized the Ser-His-Asp triad performing catalysis by bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Additionally, structural analysis of the polypropylene analog jeffamine with EstS1 revealed effective covalent binding, demonstrating its multifunctional capability. Mutation analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore the potential of EstS1 as a key tool for advancing technologies aimed at phthalate diesters biodegradation.

biophysics↗