PMID:18305117

Citation	Fukushima, T, Kitajima, T, Yamaguchi, H, Ouyang, Q, Furuhata, K, Yamamoto, H, Shida, T and Sekiguchi, J (2008) Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities. J. Biol. Chem. 283:11117-25
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."
Links	PubMed Online version:10.1074/jbc.M706626200
Keywords	Amino Acid Sequence; Aspartic Acid/chemistry; Bacillus subtilis/enzymology; Binding Sites; Cell Wall/enzymology; Endopeptidases/chemistry; Endopeptidases/physiology; Glutamic Acid/chemistry; Glycoside Hydrolases/chemistry; Glycoside Hydrolases/physiology; Hydrolases/chemistry; Hydrolases/physiology; Mass Spectrometry/methods; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Acetylmuramoyl-L-alanine Amidase/chemistry; N-Acetylmuramoyl-L-alanine Amidase/physiology; Sequence Homology, Amino Acid
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACSU:YDDH		GO:0003796: lysozyme activity	ECO:0000314:		F		The amount of released amino terminal and reduced sugar terminal groups during cell wall digestion (173.5nmol/mg and 75.0nmol/mg respectively) in CwlT-FL-His indicates muramidase/lysozyme activity (Supplement Table 3).	complete CACAO 10903
BACSU:YDDH		GO:0004175: endopeptidase activity	ECO:0000314:		F		The amount of released amino terminal and reduced sugar terminal groups during cell wall digestion (173.5nmol/mg and 75.0nmol/mg respectively) in CwlT-FL-His indicates endopeptidase activity (Supplement Table 3)	complete CACAO 10904
BACSU:YDDH	enables	GO:0004175: endopeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
BACSU:YDDH	enables	GO:0003796: lysozyme activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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