Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.
The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.
Bolhuis
A
A
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands.
Tjalsma
H
H
Stephenson
K
K
Harwood
C R
CR
Venema
G
G
Bron
S
S
van Dijl
J M
JM
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins; D002384:Catalysis; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D008565:Membrane Proteins; D009154:Mutation; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D013696:Temperature; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C104015:WprA protein, Bacillus subtilis; C026515:type I signal peptidase