Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis.
The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metalloprotease SepP1, was cloned into pT181mcs. DNA sequencing revealed an open reading frame of 1,521 nucleotides encoding a 507-amino-acid protein with an M(r) of 55,819. The sepA-containing DNA fragment did not hybridize with Staphylococcus hyicus or Staphylococcus carnosus DNA. Expression of sepA in the protease-negative S. carnosus (pT181mcsP1) resulted in overproduction of a 33-kDa protease found in the culture medium. The first 15 N-terminal amino acids of the partially purified protease completely matched the deduced DNA sequence starting at GCA (Ala-208). This finding indicated that SepP1 is synthesized as a preproenzyme with a 28-amino-acid signal peptide, a 179-amino-acid hydrophilic pro region, and a 300-amino-acid extracellular mature form with a calculated M(r) of 32,739. In activity staining, the mature protease prepared from S. carnosus (pT181mcsP1) corresponded to the extracellular S. epidermidis Tü3298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Bacillus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.
Teufel
P
P
Mikrobielle Genetik, Universität Tübingen, Germany.
Götz
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000201:enzymology / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D011480:Protease Inhibitors; D010450:Endopeptidases; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C081903:SepA protein, Staphylococcus epidermidis