title: Characterization of new L,D-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis.
abstract: Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic L,D-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a D-alanyl-D-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The beta-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-DeltaCwlK) was produced in Escherichia coli and purified on a nickel column. The h-DeltaCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-DeltaCwlK were pH 6.5, 37 degrees C, and 0 M, respectively. Interestingly, h-DeltaCwlK could hydrolyze the linkage of L-alanine-D-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of D-alanine-D-alanine, suggesting that CwlK is an L,D-endopeptidase not a D,D-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an L,D-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA.
authors.affiliation: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano, 386-8567, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fukushima
authors.forename: Yang
authors.initials: Y
authors.lastname: Yao
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kitajima
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Junichi
authors.initials: J
authors.lastname: Sekiguchi
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D008954:Models, Biological; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D010457:Peptidoglycan / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; C051467:peptidoglycan endopeptidase; D010450:Endopeptidases