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Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV
- Source :
- The Journal of biological chemistry. 276(30)
- Publication Year :
- 2001
-
Abstract
- The Bacillus subtilis CwlC and theBacillus 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.
- Subjects :
- Time Factors
DNA Mutational Analysis
Molecular Sequence Data
Glutamic Acid
Bacillus
Bacillus subtilis
Peptidoglycan
Biochemistry
Catalysis
Amidase
Bacterial Proteins
Cell Wall
Catalytic Domain
Cations
Amidase activity
Point Mutation
Magnesium
Amino Acid Sequence
Amino Acids
Molecular Biology
Alanine
chemistry.chemical_classification
Aspartic Acid
Manganese
biology
Sequence Homology, Amino Acid
C-terminus
Circular Dichroism
Cell Biology
Glutamic acid
Cobalt
N-Acetylmuramoyl-L-alanine Amidase
biology.organism_classification
Amino acid
Protein Structure, Tertiary
N-terminus
Zinc
chemistry
Mutagenesis, Site-Directed
Calcium
Electrophoresis, Polyacrylamide Gel
Gene Deletion
Plasmids
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 276
- Issue :
- 30
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....ef038354a509f6936b672337a97d3d46