Involvement of propeptides in formation of catalytically active metalloproteinase from Thermoactinomyces sp.

Dina Safina, Lola Rafieva, Ilya Demidyuk, Eugene Gasanov, Galina Chestukhina, Sergey Kostrov

Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov sq. 2, Moscow, 123182 Russia. nauruz@mail.ru

Protein and peptide letters 2011 Nov

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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.

Citation

Dina Safina, Lola Rafieva, Ilya Demidyuk, Eugene Gasanov, Galina Chestukhina, Sergey Kostrov. Involvement of propeptides in formation of catalytically active metalloproteinase from Thermoactinomyces sp. Protein and peptide letters. 2011 Nov;18(11):1119-25