Full Activation of Enterococcus faecalis Gelatinase by a C-Terminal Proteolytic Cleavage

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Journal of bacteriology (Impact Factor: 2.81). 01/2008; 189(24):8835-43. DOI: 10.1128/JB.01311-07
Source: PubMed


Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their
multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation.
Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized
as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the
molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular
mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously
identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing
event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal
deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305
and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members
of the M4 family of proteases of gram-positive bacteria.

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Available from: Vinai Chittezham Thomas, Jul 17, 2014
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    • "GelE was observed on 2D gels within the 31–44 kDa region. Significant processing of GelE via C-terminal proteolytic cleavage is required for its activation [32] and the presence of GelE spots in this region with minor variations in the pI, indicates post-translational processing of GelE such as charge changes. SprE was identified from seven spots on 2D SDS-PAGE gels, which could be due to GelE-dependent proteolysis or SprE-catalysed self-cleavage [33]. "
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