Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases Cw1C and Cw1V

The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwIV a re the cell wall lytic N-acetylmuramoyl-L-alanine amidases in the CwlB (Lyt C) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extende d slightly toward the N terminus, the amidase activity was entirely lost. F urther, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the tru ncated CwIV (CwlVt), is a catalytic domain. Site-directed mutagenesis was p erformed on 20 highly conserved amino acid residues within the catalytic do main of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substit ution of the two glutamic acid residues (E26Q and E142Q) of the truncated C wIV (CwIV1), which corresponded to Glu-24 and Glu-141 of CwIC, was critical to the amidase activity. The EDTA-treated CwIV1. did not have amidase acti vity. The amidase activity of the EDTA-treated CwIV1 was restored by the ad dition of Zn2+, Mn2+, and Co2+ but not by the addition of Me2+ and Ca2+. Th ese results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.