title: Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities.
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."
authors.affiliation: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fukushima
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kitajima
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Qin
authors.initials: Q
authors.lastname: Ouyang
authors.forename: Kazumi
authors.initials: K
authors.lastname: Furuhata
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Toshio
authors.initials: T
authors.lastname: Shida
authors.forename: Junichi
authors.initials: J
authors.lastname: Sekiguchi
mesh_terms: 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
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D006867:Hydrolases; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase; D010450:Endopeptidases; D009238:N-Acetylmuramoyl-L-alanine Amidase