Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor.
Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'.
Taguchi
S
S
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Suzuki
M
M
Kojima
S
S
Miura
K
K
Momose
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000494:pharmacology*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D012697:Serine Endopeptidases