Assessing the role of zinc in the structure-stability-activity paradigm of bacteriophage T7 endolysin

Abstract

Inorganic cofactors, such as zinc, contribute to enzymatic reactions, protein folding, and enzyme stability. T7 endolysin or T7 lysozyme (T7L), a zinc-dependent amidase involved in the lysis of bacterial cell walls, has great potential for applications in biotechnology and medicine. The present study provides an in-depth analysis of the structure stability and catalytic features of T7L, focusing on the comparison of its Apo and Holo forms using turbidimetric assay, circular dichroism, fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopic methods and molecular dynamics simulations. T7L Apo and T7L Holo forms showed moderate structural differences in the secondary structure; hydrophobic exposure was observed at the tertiary level. Chemical denaturation studies indicated higher stability in T7L Holo compared to the Apo form. The pH-dependent NMR analyses implied the protective role of zinc in the Holo form at lower pH levels, down to pH 5. The turbidimetric assay further revealed the affirmative role of zinc as a cofactor for its amidase activity. Molecular dynamics simulation studies further ally with these results, demonstrating the secondary conformational stability of T7L Holo compared to the Apo form. T7L Holo exhibits dynamic interactions with the solvent water, underlining the flexible arrangements of bonds that drive the enhanced catalytic function of T7L Holo. These results provide insights into protein enzymology, emphasizing the crucial influence of cofactors on enzymatic reactions and protein stability. The distinct features of structure-stability-activity between Apo and Holo forms of T7L under various conditions pave the way for the development of strategies based on T7 endolysin to combat microbial resistance.