Physical requirements for in vitro processing of the Streptomyces lividans signal peptidases.
The Gram-positive eubacterium Streptomyces lividans contains four chromosomally encoded type I signal peptidases, SipW, SipX, SipY and SipZ, of which all but SipW have an unusual C-terminal membrane anchor. For in vitro characterisation of these signal peptidases, the S. lividans sip genes were expressed in Escherichia coli and the corresponding proteins were purified. The four enzymes had an optimum activity at an alkaline pH, notably pH 8-9 for SipW and SipY and pH 10-11 for SipX and SipZ. In contrast to SipW, the in vitro activities of SipX, SipY and SipZ significantly increased in the presence of detergent. Since none of the S. lividans Sip proteins contains the hydrophobic beta-barrel domain, which in E. coli LepB was proven to be requisite for detergent-dependent in vitro activity, we assume that for detergent dependence, the C-terminal transmembrane anchor can partly substitute for this domain. Finally, all Sip proteins were stimulated by added phospholipids, which strongly suggests that phospholipids play an important role in the catalytic mechanism.
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000, Leuven, Belgium.
Nick
N
Geukens
Elke
E
Lammertyn
Lieve
L
Van Mellaert
Yves
Y
Engelborghs
Rafael P
RP
Mellado
Jozef
J
Anné
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*
D017931:DNA Primers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase