title: Involvement of propeptides in formation of catalytically active metalloproteinase from Thermoactinomyces sp.
abstract: The metalloproteinase from Thermoactinomyces sp. 27a (Mpr) represents secretory thermolysin-like metalloproteinases of the M4 family. The Thermoactinomyces enzyme is synthesized as a precursor consisting of a signal peptide, N-terminal propeptide, mature region, and C-terminal propeptide. The functional molecule lacks the signal peptide, N-terminal propeptide, and C-terminal propeptide, which indicates the processing of these regions. Until now, it remained unclear if the N-terminal propeptide is involved in the formation and functioning of Mpr, and the role of the C-terminal propeptide was also unclear. In this work, a Bacillus subtilis AJ73 strain expressing Mpr without the C-terminal propeptide- encoding region being involved has been obtained. The absence of the Mpr C-terminal propeptide had no significant effect on the formation of the functional molecule and did not interfere with the protease secretion in B. subtilis AJ73 cells. Strains producing the N-terminal propeptide, mature region, and mature region covalently bound to the N-terminal propeptide were generated from Escherichia coli BL-21(DE3) cells. Functionally active Mpr forms could be produced only in the presence of the N-terminal propeptide, either covalently bound to the mature region (in cis) or as a separate molecule (in trans). Thus, the Mpr three-dimensional structure is formed according to the propeptide-assisted mechanism with no requirement of a covalent bond between the N-terminal propeptide and mature region. Moreover, Mpr variants generated in cis and in trans differed in their specificity for certain synthetic substrates.
authors.affiliation: Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov sq. 2, Moscow, 123182 Russia. nauruz@mail.ru
authors.forename: Dina
authors.initials: D
authors.lastname: Safina
authors.forename: Lola
authors.initials: L
authors.lastname: Rafieva
authors.forename: Ilya
authors.initials: I
authors.lastname: Demidyuk
authors.forename: Eugene
authors.initials: E
authors.lastname: Gasanov
authors.forename: Galina
authors.initials: G
authors.lastname: Chestukhina
authors.forename: Sergey
authors.initials: S
authors.lastname: Kostrov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D055162:Biocatalysis; D002417:Cattle; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020673:Protein Renaturation; D013379:Substrate Specificity; D056509:Thermoactinomyces / Q000201:enzymology*
chemical_list: D004792:Enzyme Precursors; D010446:Peptide Fragments; D045726:Metalloproteases