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J. Biol. Chem., Vol. 283, Issue 17, 11117-11125, April 25, 2008

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Identification and Characterization of Novel Cell Wall Hydrolase CwlT

A TWO-DOMAIN AUTOLYSIN EXHIBITING N-ACETYLMURAMIDASE AND DL-ENDOPEPTIDASE ACTIVITIES^*

Tatsuya Fukushima¹, Toshihiko Kitajima¹, Hiroyuki Yamaguchi, Qin Ouyang, Kazumi Furuhata, Hiroki Yamamoto, Toshio Shida, and Junichi Sekiguchi²

From the Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, and Division of Gene Research, Department of Life Sciences, Research Center for Human and Environmental Sciences, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan

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--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."

Received for publication, August 9, 2007 , and in revised form, February 4, 2008.

^* This work was supported in part by grants-in-aid for scientific research for the 21st Century and Global COE Programs and Grants-in-aid for Scientific Research (B) 16380059 and 19380047 (to J. S.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–3 and Figs. 1–3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 81-268-21-5344; Fax: 81-268-21-5345; E-mail: jsekigu{at}shinshu-u.ac.jp.

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