Detection of large COOH-terminal domains processed from the precursor of Serratia marcescens serine protease in the outer membrane of Escherichia coli.
The Serratia marcescens serine protease gene encoding a 1,045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact precursor but also two proteins, C-1 (40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.
Shikata
S
S
Department of Agricultural Chemistry, University of Tokyo.
Shimada
K
K
Kataoka
H
H
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004269:DNA, Bacterial / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D004792:Enzyme Precursors; D012697:Serine Endopeptidases