Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities.
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."
Fukushima
Tatsuya
T
Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
Kitajima
Toshihiko
T
Yamaguchi
Hiroyuki
H
Ouyang
Qin
Q
Furuhata
Kazumi
K
Yamamoto
Hiroki
H
Shida
Toshio
T
Sekiguchi
Junichi
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002473:Cell Wall / Q000201:enzymology*; D010450:Endopeptidases / Q000737:chemistry / Q000502:physiology*; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000502:physiology*; D006867:Hydrolases / Q000737:chemistry / Q000502:physiology*; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid
D001224:Aspartic Acid; D018698:Glutamic Acid; D006867:Hydrolases; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase; D010450:Endopeptidases; D009238:N-Acetylmuramoyl-L-alanine Amidase