title: Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus.
abstract: The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49,698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38,394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55 degrees C and pH 7.4-8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn2+ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn2+, indicated that Zn2+ is the catalytic ion. Ca2+ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.
authors.affiliation: Universität Tübingen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Ayora
authors.forename: F
authors.initials: F
authors.lastname: Götz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008666:Metalloendopeptidases; C086144:ShpI metalloprotease