SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoatractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.
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