Abstract
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.
Highlights
The cell wall of Bacillus subtilis is ϳ25–30 nm thick and contains roughly 50% by weight peptidoglycan
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 of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain
To determine the catalytic amino acid residues, sitedirected mutagenesis was performed on 20 amino acid residues within the N-terminal 175 amino acids of the CwlC amidase and on two amino acid residues of the truncated CwlV amidase (CwlV1)
Summary
Enzymes and Chemicals—Restriction endonucleases and a ligation kit were purchased from Takara Shuzo (Kyoto, Japan). The antisense primer was 5ЈGCTGCCTGCAGCTATGATTCTAGGATCACAATAGC-3Ј (the complimentary sequence of the cwlC sequence is italicized, the complimentary sequence of the TAG termination codon is boldfaced, and the PstI site is underlined). The mutagenized sense primer was 5Ј-CCGCGGAATTCTATGAAGGTTGTTGTTATTGATGCTGG-3Ј (the cwlV sequence is italicized, the initiation codon is boldfaced, and the EcoRI site is underlined). The antisense primer was 5Ј-GCTGCCTGCAGTTATTTTACGTCGAGATACTCTGT-3Ј (the complimentary sequence of the cwlV sequence is italicized, the complimentary sequence of the TAA termination codon is boldfaced, and the PstI site is underlined). The solution was applied onto an SP-Sepharose column (Amersham Pharmacia Biotech), and proteins were eluted with a linear gradient from 0.02 to 1 M NaCl. The fractions containing CwlV1 were collected and dialyzed against the dialysis buffer (20 mM potassium phosphate, pH 7.0). Proteins on a gel were renatured by treatment with 0.1 M Tris-HCl (pH 8.0)
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