Detergent-independent in vitro activity of a truncated Bacillus signal peptidase.
The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory pre-proteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Maarten L
ML
van Roosmalen
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Jan D H
JDH
Jongbloed
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Anne de
A
Jong
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Jaap
J
van Eerden
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Gerard
G
Venema
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Sierd
S
Bron
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands1.
Jan
J
Maarten van Dijl
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D003902:Detergents; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase