In Vitro Processing of Glutamyl Endopeptidase Proenzymes from Enterococcus faecalis and Importance of N-terminal Residue in Enzyme Catalysis

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Glutamyl endopeptidase from Enterococcus faecalis, designated SprE, is one of the important virulence factors secreted as zymogen. In the present study we expressed recombinant SprE proenzyme (pro-SprE) in Escherichia coli and investigated the in vitro processing to mature SprE. It was found that trypsin could efficiently produce the active form of SprE with the N-terminus Ser1 through cleavage between Arg-1 and Ser1 bond, which was subsequently auto-degraded into inactive species through the cleavage at the Glu6-Asp7 and Glu11-Val12 bonds. Although thermolysin could produce SprE with the N-terminus Leu2, but possessed no proteolytic activity. In contrast to the absolute requirement of the N-terminal Val1 in staphylococcal glutamyl endopeptidases, the N-terminal Ser1 of mature SprE could be substituted by other amino acids despite that Ser showed the maximal activity. Substitution of penultimate Leu2 of SprE to Val2 also reduced the activity to 40% of the wild type. Taken together, we conclude that pro-SprE was converted to mature form with the N-terminus Ser1 by a protease with specificity of trypsin and the length of the N-terminal region rather than specific residue is absolutely required for enzyme activity.