Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

Fukushima, Tatsuya; Yao, Yang; Kitajima, Toshihiko; Yamamoto, Hiroki; Sekiguchi, Junichi

doi:10.1007/s00438-007-0255-8

Cited by 44 publications

(39 citation statements)

References 38 publications

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“…CwlT-FL-His-Determination of the optimum pH values and temperatures of the truncated CwlT proteins was performed as described previously (3,13,18). One unit of hydrolase activity was defined as the amount of enzyme necessary to decrease the absorbance at 540 nm by 0.001 in 1 min (3,13,18).…”

Section: Determination Of the Optimum Ph Values And Temperatures Of Cmentioning

confidence: 99%

“…One unit of hydrolase activity was defined as the amount of enzyme necessary to decrease the absorbance at 540 nm by 0.001 in 1 min (3,13,18).…”

Section: Determination Of the Optimum Ph Values And Temperatures Of Cmentioning

confidence: 99%

“…For Bacillus subtilis, more than 30 candidate peptidoglycan hydrolases are proposed on the basis of amino acid sequence similarity (1), and these enzymes are important in various cellular processes during vegetative growth, sporulation, and germination (1,2). Several groups of peptidoglycan hydrolases are enzymatically identified as follows: N-acetylglucosaminidases (digesting GlcNAc-MurNAc 3 linkage), N-acetylmuramoyl-Lalanine amidases (digesting MurNAc-L-alanine linkage), DL-endopeptidases (digesting D-glutamic acid-meso-A 2 pm linkage) (1,2), and LD-endopeptidase (digesting L-alanine-D-glutamic acid linkage) (3). However, the characterization of the group of DD-endopeptidase (digesting the cross-linked D-alanine-meso-A 2 pm linkage) has not been reported in B. subtilis.…”

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confidence: 99%

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Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Fukushima

Kitajima

Yamaguchi

et al. 2008

Journal of Biological Chemistry

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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."Many microorganisms have peptidoglycan as a major component of the cell wall, which consists of glycan strands crosslinked by peptides. Bacteria have various cell wall lytic enzymes. For Bacillus subtilis, more than 30 candidate peptidoglycan hydrolases are proposed on the basis of amino acid sequence similarity (1), and these enzymes are important in various cellular processes during vegetative growth, sporulation, and germination (1, 2). Several groups of peptidoglycan hydrolases are enzymatically identified as follows: N-acetylglucosaminidases (digesting GlcNAc-MurNAc 3 linkage), N-acetylmuramoyl-Lalanine amidases (digesting MurNAc-L-alanine linkage), DL-endopeptidases (digesting D-glutamic acid-meso-A 2 pm linkage) (1, 2), and LD-endopeptidase (digesting L-alanine-D-glutamic acid linkage) (3). However, the characterization of the group of DD-endopeptidase (digesting the cross-linked D-alanine-meso-A 2 pm linkage) has not been reported in B. subtilis. Interestingly, the groups of muramidase and lytic transglycosylase (digesting MurNAc-GlcNAc linkage) in B. subtilis are still not characterized, even many hydrolases in those groups, including hen egg white lysozyme, have already been identified.Previously Atrih et al. (4) described that the vegetative peptidoglycan in B. subtilis includes (136)-anhydro-N-acetylmuramic acid. Thus, it is possible that lytic transglycosylase, which digests MurNAc-GlcNAc linkage with synthesis of a 1,6-anhydro bond in the N-acetylmuramic acid (5), hydrolyzes the vegetative peptid...

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Section: Determination Of the Optimum Ph Values And Temperatures Of Cmentioning

confidence: 99%

“…One unit of hydrolase activity was defined as the amount of enzyme necessary to decrease the absorbance at 540 nm by 0.001 in 1 min (3,13,18).…”

Section: Determination Of the Optimum Ph Values And Temperatures Of Cmentioning

confidence: 99%

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confidence: 99%

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Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Fukushima

Kitajima

Yamaguchi

et al. 2008

Journal of Biological Chemistry

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“…Overexpression and Purification of CwlH, CwlK, and LytF (CwlE)-CwlH (L-alanine amidase), CwlK (LD-endopeptidase), and LytF (CwlE) (DL-endopeptidase) were overexpressed and purified from E. coli cells as described previously (20,22,23).…”

Section: Sds-page and Purification Of Cell Wall And Peptidoglycan Fromentioning

confidence: 99%

Identification and Characterization of a Novel Polysaccharide Deacetylase C (PdaC) from Bacillus subtilis

Kobayashi

Sudiarta

Kodama

et al. 2012

Journal of Biological Chemistry

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“…Comparison of the hydrolytic activities of the proteins were obtained by measurement of the cell wall densities using a spectrophotometer (V-560, JASCO), as described previously (11,12). The hydrolytic reactions were performed in 50 mM MES-NaOH buffer, pH 5, at 37°C for CwlP-SLT and in 50 mM MOPSNaOH buffer, pH 7.5, at 40°C for CwlP-M23.…”

Section: Construction Of Plasmids For Overexpression Of the Peptidasementioning

confidence: 99%

Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase

Sudiarta

Fukushima

Sekiguchi

2010

Journal of Biological Chemistry

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For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-␤ prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave D-Ala-diaminopimelic acid cross-linkage, a property associated with DD-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn 2؉ -independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn 2؉ dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.The cycle of bacteriophage infection of microorganisms comprises adsorption, insertion of nucleic acids, production of bacteriophage nucleic acids and proteins, and finally, host cell lysis. The infection cycle follows a highly ordered sequence of events where cell wall hydrolases, encoded in bacteriophage genomes, are involved in adsorption to cell walls (first infection cycle) and host cell lysis (final infection cycle) (1).One of the best studied Gram-positive bacteria, Bacillus subtilis, has prophage-like elements including PBSX, skin, and SP-␤ (2). The SP-␤ prophage is the largest prophage-like element in B. subtilis (2). Inspection of the BSORF database indicates that the SP-␤ prophage chromosomal region contains 184 genes and one cell wall hydrolase called BlyA, which has been identified as an L-alanine amidase (3). Interestingly, the Pfam database predicts that another gene product, YomI (renamed CwlP (cell wall lytic enzyme related to phage)), has two cell wall hydrolase domains, a soluble lytic transglycosylase (SLT) 3 and peptidase M23 (Fig. 1). However, the function of the two domains (SLT and peptidase M23) remains unclear. BLAST searches of the BSORF and Pfam databases indicate that the SLT domain contains a soluble lytic transglycosylase and a muramidase, although the domain name is annotated "SLT." At present, lytic transglycosylases and muramidases cannot be differentiated using amino acid sequence similarity. The peptidase M23 domain has hydrolytic activity, enabling it to digest the Gly-Gly bond. It is also known that LytM in Staphylococcus aureus has a peptidase M2...

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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

Cited by 44 publications

References 38 publications

Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Identification and Characterization of a Novel Polysaccharide Deacetylase C (PdaC) from Bacillus subtilis

Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase

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