Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants

The type I signal peptidase SipS of Bacillus subtilis is of major importanc e for the processing of secretory precursor proteins. In the present studie s, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of th ese mutants, L74A and Y81A, are structurally stable but strongly impaired i n catalytic activity at 48 degrees C, showing the (unprecedented) involveme nt of the conserved leucine 74 and tyrosine 81 residues in the catalytic re action of type I signal peptidases. This conclusion is supported by the cry stal structure of the homologous signal peptidase of Escherichia coli (Paet zel, M., Dalbey, R. E., and Strynadka, N, C, J, (1998) Nature 396, 188-190) , In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactiva ted by proteolytic degradation, indicating that the conserved arginine 84 a nd aspartic acid 146 residues are required to obtain a protease-resistant c onformation. 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 h as a large extracytoplasmic domain. As WprA was not involved in the degrada tion of the SipS mutant proteins R84A and R84H, we conclude that multiple p roteases are responsible for the thermal inactivation of temperature-sensit ive SipS mutants.