Integrative In Silico and Experimental Characterization of Endolysin LysPALS22: Structural Diversity, Ligand Binding...

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Antimicrobial Research

Background:

• Endolysins, phage-derived enzymes, are promising antimicrobials against resistant bacteria.
• Novel endolysins require detailed characterization for therapeutic development.
• Understanding enzyme structure-function relationships is crucial for optimizing antimicrobial activity.

Purpose of the Study:

• To conduct an integrative in silico and experimental characterization of the novel endolysin LysPALS22.
• To elucidate the structural features, substrate-binding mechanisms, and recombinant expression of LysPALS22.
• To establish a foundation for developing LysPALS22 as a novel antimicrobial agent.

Main Methods:

• Multiple sequence alignment and phylogenetic analysis of endolysin sequences.
• Generation and validation of 3D structural models using Swiss-Model, EBI-EMBL, and AlphaFold Colab.
• Molecular docking and dynamics simulations to analyze enzyme-ligand interactions.
• Heterologous expression of LysPALS22 in Escherichia coli and Pichia pastoris, followed by protein analysis.

Main Results:

• Phylogenetic analysis revealed conserved N-terminal catalytic and binding domains in endolysins.
• Swiss-Model provided the highest quality 3D structure for LysPALS22, showing >90% residues in favored conformations.
• Molecular docking identified N-Acetylmuramic Acid-L-Alanine as a high-affinity ligand (-7.3 kcal/mol) with specific hydrogen bonding to ASP46 and TYR61.
• Molecular dynamics simulations confirmed the stability of the LysPALS22-ligand complex.
• Pichia pastoris expression yielded higher volumetric yields (1.56 mg/mL) and showed glycosylation compared to E. coli (1.31 mg/mL).

Conclusions:

• LysPALS22 possesses conserved enzymatic features and specific substrate recognition capabilities.
• The validated structural and interaction data provide a strong basis for LysPALS22's potential as an antimicrobial.
• Pichia pastoris is a suitable host for high-yield recombinant production of LysPALS22, supporting further development.