期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 398, 期 4, 页码 507-517出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.03.033
关键词
autolysin; lysostaphin; endopeptidase; peptidoglycan crossbridge; Enterococcus faecalis
资金
- Agence Nationale de la Recherche [ANR-07-MIME-020]
Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl- glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpA(C), comprising residues 1374-1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpA(C) cleaves the D-Ala-L-Ala bond formed by the D,D-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic D was identified as the minimum substrate of EnpA(C) indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpA(C) to assess enzyme specificity. EnpA(C) did not cleave direct cross-links, but tolerated extensive variation in crossbridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpA(C), which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family. (C) 2010 Elsevier Ltd. All rights reserved.
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