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Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

Abstract : The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.
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Contributor : Maxime Lecerf <>
Submitted on : Tuesday, June 19, 2007 - 6:13:00 PM
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Catherine Eckert, Maxime Lecerf, Lionel Dubost, Michel Arthur, Stéphane Mesnage. Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.. Journal of Bacteriology, American Society for Microbiology, 2006, 188 (24), pp.8513-9. ⟨10.1128/JB.01145-06⟩. ⟨inserm-00156004⟩



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