Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme

Abstract The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

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Specific excretion into the medium of a serine protease from Serratia marcescens.

Agricultural and Biological Chemistry ◽

10.1271/bbb1961.54.2763 ◽

1990 ◽

Vol 54 (10) ◽

pp. 2763-2765 ◽

Cited By ~ 7

Author(s):

Hiroyuki MIYAZAKI ◽

Noboru YANAGIDA ◽

Sueharu HORINOUCHI ◽

Teruhiko BEPPU

Keyword(s):

Serratia Marcescens ◽

Serine Protease

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Two Genes Encoding Serine Protease Homologues in Serratia marcescens and Characterization of Their Products in Escherichia coli

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a021672 ◽

1997 ◽

Vol 121 (5) ◽

pp. 902-913 ◽

Cited By ~ 15

Author(s):

Y. Ohnishi ◽

T. Beppu ◽

S. Horinouchi

Keyword(s):

Escherichia Coli ◽

Serratia Marcescens ◽

Serine Protease ◽

Genes Encoding

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Characterization of Secretory Intermediates of Serratia marcescens Serine Protease Produced during Its Extracellular Secretion from Escherichia coli Cells

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a124244 ◽

1993 ◽

Vol 114 (5) ◽

pp. 723-731 ◽

Cited By ~ 14

Author(s):

Shitsuw Shikata ◽

Kohei Shimada ◽

Yasuo Ohnishi ◽

Sueharu Horinouchi ◽

Teruhiko Beppu

Keyword(s):

Escherichia Coli ◽

Serratia Marcescens ◽

Serine Protease ◽

Extracellular Secretion ◽

Escherichia Coli Cells

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Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli.

Journal of Bacteriology ◽

10.1128/jb.171.12.6566-6572.1989 ◽

1989 ◽

Vol 171 (12) ◽

pp. 6566-6572 ◽

Cited By ~ 33

Author(s):

H Miyazaki ◽

N Yanagida ◽

S Horinouchi ◽

T Beppu

Keyword(s):

Escherichia Coli ◽

Serratia Marcescens ◽

Outer Membrane ◽

Serine Protease

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Characterization of the gene encoding the glutamic-acid-specific protease of Streptomyces griseus

Biochemistry and Cell Biology ◽

10.1139/o93-067 ◽

1993 ◽

Vol 71 (9-10) ◽

pp. 454-461 ◽

Cited By ~ 11

Author(s):

Sachdev S. Sidhu ◽

Gabriel B. Kalmar ◽

Thor J. Borgford

Keyword(s):

Glutamic Acid ◽

Serine Protease ◽

Streptomyces Griseus ◽

Gene Encoding ◽

Lysobacter Enzymogenes ◽

Glutamic Acid Residue ◽

Specific Protease ◽

Mature Enzyme ◽

Additional Segment ◽

Pro Region

The complete gene sequence (sprE) for the glutamic-acid-specific serine protease (SGPE) of the gram-positive bacterium Streptomyces griseus is reported. The sprE gene encodes a 355 amino acid pre–pro–mature enzyme. The presence of a glutamic acid residue at the junction of the pro and mature segments of the protein suggests that the enzyme is self-processing. SGPE was found to have extensive homology with the S. griseus proteases A and B. However, there is an additional segment to the pro region of SGPE, lacking in proteases A and B, which has significant homology to the pro region of the α-lytic protease of the gram-negative bacterium Lysobacter enzymogenes. Expression of recombinant SGPE in Bacillus subtilis is also reported, and the enzyme is shown to be self-processing.Key words: serine protease, maturation, expression, propeptide.

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