Abstract
A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."
Highlights
Many microorganisms have peptidoglycan as a major component of the cell wall, which consists of glycan strands crosslinked by peptides
We found that the C-terminal domain is a DLendopeptidase that hydrolyzes the linkage of D-␥-glutamylmeso-diaminopimelic acid of peptidoglycan and that the N-terminal domain is an N-acetylmuramidase, which digests the linkage of MurNAc-GlcNAc even though it is predicted as an soluble lytic transglycosylase (SLT) domain
Amino Acid Sequence of CwlT—It is predicted that CwlT has two domains associated with cell wall hydrolase activity
Summary
A TWO-DOMAIN AUTOLYSIN EXHIBITING N-ACETYLMURAMIDASE AND DL-ENDOPEPTIDASE ACTIVITIES*□S. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to DL-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents “a new category of cell wall hydrolases.”. The groups of muramidase and lytic transglycosylase (digesting MurNAc-GlcNAc linkage) in B. subtilis are still not characterized, even many hydrolases in those groups, including hen egg white lysozyme, have already been identified. It is possible that lytic transglycosylase, which digests MurNAc-GlcNAc linkage with synthesis of a 1,6-anhydro bond in the N-acetylmuramic acid [5], hydrolyzes the vegetative peptidoglycan. We report that the active sites for hydrolysis of the N-terminal domain are Glu-87 and Asp-94
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