JB Accepts, published online ahead of print on 5 October 2007

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J. Bacteriol. doi:10.1128/JB.01311-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Maria Florencia Del Papa, Lynn Hancock, Vinai Thomas, and Marta Perego^*

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; Division of Biology, Kansas State University, Manhattan, KS 66506-4901

^* To whom correspondence should be addressed. Email: mperego{at}scripps.edu.

Enterococci account for nearly 10 % of all nosocomial infections and constitute a significant treatment challenge due to their multi-drug resistance properties. One of the well studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which was 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 the mature enzyme. We determined that the molecular mass of the mature protein isolated from the culture supernatant was 33,030 Da, which differed from the predicted mass of 34,570 Da by over 1,500 Daltons. Using N-terminal sequencing we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500 Dalton mass difference resulted from a C-terminal processing event. By means of site-directed mutants within a predicted C-terminal processing site and C-terminal deletion mutants fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305, and 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 Gram-positive members of the M4 family of proteases.

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