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Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants

Lookup NU author(s): Professor Colin Harwood, Dr Sierd Bron

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Abstract

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°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.


Publication metadata

Author(s): Harwood CR; Bron S; Bolhuis A; Tjalsma H; Stephenson K; Venema G; Van Dijl JM

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 1999

Volume: 274

Issue: 22

Pages: 15865-15868

Print publication date: 28/05/1999

ISSN (print): 0021-9258

ISSN (electronic):

Publisher: American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.274.22.15865

DOI: 10.1074/jbc.274.22.15865

PubMed id: 10336490


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