Genetic evidence that antibacterial activity of lysozyme is independent ofits catalytic function

A catalytically inactive mutant of hen egg white lysozyme was constructed b y site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic re sidue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2. Western blot analysis indicated that the mutation did not affect se cretion of the D52S-Lz lysozyme into the medium of the expressing Saccharom yces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence s pectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the cata lytic activity of lysozyme. Antimicrobial tests against Staphylococcus aure us and Bacillus subtilis revealed that the catalytically inactive D52S-Lz w as as bactericidal as the Wt-Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme. The binding affinity of D52S-Lz to the iso lated peptidoglycan of S. aureus was unaffected. Our results provide the fi rst demonstration of direct genetic evidence that the antimicrobial activit y of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors. (C) 2001 Federation of European Biochemical Societies. Published b y Elsevier Science B.V. All rights reserved.