In vitro and in vivo self‐cleavage of Streptococcus pneumoniae signal peptidase I

We have previously demonstrated that Streptococcus pneumoniae signal peptidase (SPase) I catalyzes a self‐cleavage to result in a truncated product, SPase37–204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. Bacteriol. 183 , 621–627]. In this study, we investigated the effect of phospholipid on invitro self‐cleavage of S. pneumoniae SPase I. In the presence of phospholipid, the self‐cleavage predominantly occurred at one cleavage site between Gly36–His37, whereas the self‐cleavage occurred at multiple sites in the absence of phospholipid, and two additional self‐cleavage sites, Ala65–His66 and Ala143–Phe144, were identified. All three self‐cleavage sites strongly resemble the signal peptide cleavage site and follow the (−1, −3) rule for SPase I recognition. Kinetic analysis demonstrated that self‐cleavage is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25‐fold higher than that in the absence of phospholipid. Biochemical analysis demonstrated that SPase37–204, the major product of the self‐cleavage totally lost activity to cleave its substrates, indicating that the self‐cleavage resulted in the inactivation of the enzyme. More importantly, the self‐cleavage was demonstrated to be happening in vivo in all the growth phases of S. pneumoniae cells. The bacterial cells keep the active SPase I at the highest level in exponential growth phase, suggesting that the self‐cleavage may play an important role in regulating the activity of the enzyme under different conditions.