Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV.
The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, 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 truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.
Shida
T
T
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
Hattori
H
H
Ise
F
F
Sekiguchi
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001407:Bacillus / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D002473:Cell Wall / Q000201:enzymology*; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D018698:Glutamic Acid / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry; D010957:Plasmids; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D002412:Cations; D010457:Peptidoglycan; D001224:Aspartic Acid; D003035:Cobalt; D018698:Glutamic Acid; D008345:Manganese; C083494:cwlC protein, Bacillus subtilis; D009238:N-Acetylmuramoyl-L-alanine Amidase; D008274:Magnesium; D015032:Zinc; D002118:Calcium