Elucidating the functional significance of catalytic and chitin-binding domains for the anti-cancer property of a bacterial endochitinase.

Chitinases are enzymes that facilitate the breakdown of chitin and also interact with carbohydrate moieties such as heparin sulphate due to structural similarity with chitin, thereby influencing cell adherence and migration. Bacterial endo-chitinase contains several domains, such as a catalytic domain, an FNIII domain, and a chitin-binding domain. Recent studies have demonstrated the anti-cancer property of catalytically active chitinases; however, the mechanism and targets have not yet been explored. Therefore, this study investigates the importance of the catalytic and chitin-binding domains of a bacterial endo-chitinase (ChiC) for the anti-cancer property by creating domain-specific mutants. Initially, an in silico study identified an evolutionarily conserved tryptophan (W300) within the catalytic cleft of ChiC, which was subjected to site-directed mutagenesis to create an inactive ChiC mutant. Similarly, another ChiC mutant was created without the chitin-binding domain. The recombinantly expressed WT and mutant ChiC proteins were utilized to analyze their effects on the cell viability, cell cycle, and migratory and invasive behaviour of MCF-7 cells. The mutants of ChiC, lacking either catalytic property or chitin-binding domain, resulted in a loss of their ability to inhibit cell proliferation and migration. These observations suggest that the catalytic and chitin-binding domains are essential for exhibiting anti-proliferative and anti-migratory effects, providing insights for future therapeutic applications.