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.
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA.
Feng
F
Zheng
Eddie L
EL
Angleton
Jin
J
Lu
Sheng-Bin
SB
Peng
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology; D013300:Streptokinase / Q000378:metabolism; D013379:Substrate Specificity
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004792:Enzyme Precursors; D008565:Membrane Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D013300:Streptokinase; D012697:Serine Endopeptidases; C026515:type I signal peptidase