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A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase
1Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
Abstract:
A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.
Insights
A novel Streptomyces caespitosus neutral proteinase inhibitor (ScNPI) was discovered. This double-headed inhibitor targets both Streptomyces caespitosus neutral proteinase (ScNP) and subtilisin BPN' through distinct reactive sites.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Metalloproteinases play crucial roles in various biological processes.
- Specific inhibitors are essential tools for studying enzyme function and regulation.
- Streptomyces species are known sources of bioactive compounds, including protease inhibitors.
Purpose of the Study:
- To isolate and characterize a novel proteinaceous inhibitor from Streptomyces sp. I-355.
- To elucidate the inhibitory mechanism and substrate specificity of the novel inhibitor.
- To identify the reactive sites responsible for inhibiting metalloproteinases and serine proteases.
Main Methods:
- Isolation and purification of the inhibitor from culture supernatant.
- Enzyme inhibition assays to determine K(i) values against ScNP and subtilisin BPN'.
- Site-directed mutagenesis to identify reactive sites and gene sequencing.
- Analysis of complex stoichiometry using analytical techniques.
Main Results:
- A novel inhibitor, ScNPI, was isolated and exhibited potent inhibition against ScNP (K(i) = 1.6 nm) and subtilisin BPN' (K(i) = 1.4 nm).
- The scnpi gene encodes a protein with high similarity to Streptomyces subtilisin inhibitor (SSI).
- Mutagenesis studies revealed distinct reactive sites for ScNP and subtilisin BPN', identifying ScNPI as a double-headed inhibitor.
- A 2:2:2 stoichiometry was observed in complexes formed by ScNPI, ScNP, and subtilisin BPN'.
Conclusions:
- ScNPI is a novel, double-headed inhibitor with dual specificities for a metalloproteinase and a serine protease.
- The inhibitor possesses distinct reactive sites, enabling simultaneous or sequential inhibition of different protease classes.
- ScNPI serves as a valuable molecular probe for investigating the roles of ScNP and subtilisin BPN' in biological systems.