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Journal of Bacteriology, January 2005, p. 266-275, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.266-275.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Diversity of a Putative Virulence Factor: Purification and Characterization of Isoforms of an Extracellular Serine Glutamyl Endopeptidase of Enterococcus faecalis with Different Enzymatic Activities

Magdalena Kawalec,1,2 Jan Potempa,3,4* Jonathan L. Moon,3 James Travis,3 and Barbara E. Murray1,5

Division of Infectious Diseases, Department of Internal Medicine,1 Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston, Texas,5 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia,3 National Institute of Public Health, Warsaw,2 Department of Microbiology, Faculty of Biotechnology, Jagiellonian University, Cracow, Poland4

Received 10 May 2004/ Accepted 21 September 2004

A previously identified gene sprE of Enterococcus faecalis strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type E. faecalis strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser–1-Leu1 and Arg230-Leu231 peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of gelE which encodes the E. faecalis gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu1-Ala237, Ser–1-Glu227, and Leu1-Glu227, were identified. As with the V8 proteinase of Staphylococcus aureus, the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and ß-chain insulin, was the Ser–1-Glu227 (–1S-SprE) isolated from TX5264; –1S-SprE, in contrast to other forms of SprE, was unstable at 37°C, apparently due to autodegradation. In conclusion, our results demonstrate that sprE encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a "superactive" form of the enzyme, –1S-SprE.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of Georgia, Life Science Bldg. A322, Athens, GA 30602. Phone: (706) 542-1713. Fax: (706) 542-3719. E-mail: potempa{at}uga.edu.

Journal of Bacteriology, January 2005, p. 266-275, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.266-275.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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  • Singh, K. V., Nallapareddy, S. R., Nannini, E. C., Murray, B. E. (2005). Fsr-Independent Production of Protease(s) May Explain the Lack of Attenuation of an Enterococcus faecalis fsr Mutant Versus a gelE-sprE Mutant in Induction of Endocarditis. Infect. Immun. 73: 4888-4894 [Abstract] [Full Text]  


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