Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function.
A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic residue 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 secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was 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 isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors.
Ibrahim
H R
HR
Department of Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. hishamri@chem.agri.kagoshima-u.ac.jp
Matsuzaki
T
T
Aoki
T
T
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D017931:DNA Primers; D008826:Microbial Sensitivity Tests; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D013211:Staphylococcus aureus / Q000187:drug effects
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D009113:Muramidase