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Journal of Bacteriology, December 2007, p. 8835-8843, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01311-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Full Activation of Enterococcus faecalis Gelatinase by a C-Terminal Proteolytic Cleavage ^{, ,}

Maria Florencia Del Papa ,^1,,¶ Lynn E. Hancock,^1,2, Vinai C. Thomas,² and Marta Perego^1*

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037,¹ Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901²

Received 13 August 2007/ Accepted 27 September 2007

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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* Corresponding author. Mailing address: Division of Cellular Biology, Mail Code MEM-116, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-7912. Fax: (858) 784-7966. E-mail: mperego{at}scripps.edu

Published ahead of print on 5 October 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

Manuscript 19055 from The Scripps Research Institute.

M.F.D.P. and L.E.H. contributed equally to this study.

^¶ Present address: Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calles 47 y 115 (1900), La Plata, BsAS, Argentina.

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Journal of Bacteriology, December 2007, p. 8835-8843, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01311-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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