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Involvement of Propeptides in Formation of Catalytically Active Metalloproteinase from Thermoactinomyces sp.

[ Vol. 18 , Issue. 11 ]

Author(s):

Dina Safina, Lola Rafieva, Ilya Demidyuk, Eugene Gasanov, Galina Chestukhina and Sergey Kostrov   Pages 1119 - 1125 ( 7 )

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, Nterminal 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.

Keywords:

C-terminal propeptide, metalloproteinase, n-terminal propeptide, substrate specificity, Mpr, AJ73, TLPs, SNMatC, Microbial strains and vectors, DNA polymerase, NMat gene, SDS, Gel permeation chromatography, SNMat q, Mpr variantC-terminal propeptide, metalloproteinase, n-terminal propeptide, substrate specificity, Mpr, AJ73, TLPs, SNMatC, Microbial strains and vectors, DNA polymerase, NMat gene, SDS, Gel permeation chromatography, SNMat q, Mpr variant

Affiliation:

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



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