Comparative studies on the primary structures and inhibitory properties of subtilisin‐trypsin inhibitors from <i>Streptomyces</i>

Taguchi, Seiichi; Kojima, Shinichi; Terabe, Mahito; Miura, Kiyonori; Momose, Haruo

doi:10.1111/j.1432-1033.1994.tb18694.x

Cited by 26 publications

(17 citation statements)

References 27 publications

(19 reference statements)

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“…SSIs modulate the activity of extracellular serine proteases, including subtilisin-type proteases, chymotrypsins, and trypsins, by direct binding (29). We also found that the SSI gene in S. griseus is a member of the AdpA regulon (unpublished data).…”

Section: Control Of Extracellular Proteases By A-factor Via Adpamentioning

confidence: 76%

Three Chymotrypsin Genes Are Members of the AdpA Regulon in the A-Factor Regulatory Cascade in Streptomyces griseus

et al. 2005

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AdpA is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for secondary metabolism and morphological differentiation. Of the five chymotrypsin-type serine protease genes, sprA, sprB, and sprD were transcribed in response to AdpA, showing that these protease genes are members of the AdpA regulon. These proteases were predicted to play the same physiological role, since these protease genes were transcribed in a similar time course during growth and the matured enzymes showed high end-to-end similarity to one another. AdpA bound two sites upstream of the sprA promoter approximately at positions ؊375 and ؊50 with respect to the transcriptional start point of sprA. Mutational analysis of the AdpA-binding sites showed that both AdpA-binding sites were essential for transcriptional activation. AdpA bound a single site at position ؊50 in front of the sprB promoter and greatly enhanced the transcription of sprB. The AdpA-binding site at position ؊40 was essential for transcription of sprD, although there was an additional AdpA-binding site at position ؊180. Most chymotrypsin activity excreted by S. griseus was attributed to SprA and SprB, because mutant ⌬sprAB, having a deletion in both sprA and sprB, lost almost all chymotrypsin activity, as did mutant ⌬adpA. Even the double mutant ⌬sprAB and triple mutant ⌬sprABD grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) is a chemical signaling molecule, or a microbial hormone, that triggers secondary metabolism and cell differentiation in Streptomyces griseus (7,8). We have revealed the A-factor regulatory cascade, through which production of almost all the secondary metabolites produced by S. griseus and formation of aerial mycelium and spores are triggered. A-factor is gradually accumulated in a growth-dependent manner, with its maximum quantity, about 25 ng/ml, at or near the decision point (1). The decision point is at the middle of the exponential growth phase (20). When the concentration of A-factor reaches a critical level, it binds ArpA, which has bound the promoter of adpA, and dissociates the ArpA from the DNA, thus inducing the transcription of adpA (7,22). The transcriptional factor AdpA activates a number of genes required for secondary metabolism and morphological differentiation. Members of the AdpA regulon include strR, the pathwayspecific transcriptional activator for streptomycin biosynthesis (30); an open reading frame encoding a probable pathwayspecific regulator for a polyketide compound (35); adsA encoding an extracytoplasmic function sigma factor of RNA polymerase essential for aerial mycelium formation (32); ssgA encoding a small acidic protein essential for spore septum formation (33); and amfR encoding a regulatory protein essential for aerial mycelium formation (34). In addition to these genes, sgmA encoding a metalloendopeptidase (12) and sprT and sprU, both e...

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Section: Control Of Extracellular Proteases By A-factor Via Adpamentioning

confidence: 76%

Three Chymotrypsin Genes Are Members of the AdpA Regulon in the A-Factor Regulatory Cascade in Streptomyces griseus

et al. 2005

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“…(16)(17)(18) and that a strong correlation exists between the structure around the reactive region of SSI-like proteins and their inhibition specificities toward proteases (19,25). In this situation, the isolation and characterization of an endogenous extracellular protease(s) acting as a true target enzyme(s) interactive with SSI would enable us to explore the physiological role and evolutionary process of the interaction system between protease and a protease inhibitor in Streptomyces spp.…”

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“…Then, an equal volume of ice-cold 0.4 M Gly-HCl (pH 2.5) was added and incubated at 4ЊC for 5 min, after which proteins were precipitated with a final concentration of 20% trichloroacetic acid. The precipitate was dissolved in 0.1% trifluoroacetic acid and subjected to reverse-phase HPLC on a C 18 column under the conditions described previously (19).…”

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Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor

et al. 1995

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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؇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 ؋ 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 as that toward an exogenous target enzyme, subtilisin BPN.Streptomyces spp. are filamentous gram-positive soil bacteria widely known as producers of a variety of hydrolytic enzymes as well as of commercially important antibiotics. A commercial crude sample, pronase (13), prepared from Streptomyces griseus is a mixture of several extracellular proteolytic enzymes and has been widely used in biological studies for a long time. Serine proteases produced by Streptomyces lactamdurans (5) and Streptomyces peucetius (4) were reported to coordinately regulate the cellular protein turnover associated with secondary metabolism and morphogenesis. This organism is also well known to produce protease inhibitors, including low-molecular-mass (26) and proteinaceous (6) compounds. A protease inhibitor might be considered a modulator molecule for the proteolytic activities of some enzymes involved in physiological processes, such as cell differentiation and secondary metabolism, as suggested by Aoyagi (1). However, the biological roles of the interactions between protease and protease inhibitors in the producer strains have not been well identified. Streptomyces su...

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“…SSI and SSIlike protease inhibitors (SIL) produced by a variety of Streptomyces species and its related species form a large SSI family. [3][4][5] Wide distribution of SSI and SIL in actinobacteria suggests an important common role of these inhibitors in the producers. Although the biochemistry of SSI, including their structure-function relationships on the basis of X-ray crystallography of SSI and an SSI-subtilisin BPN 0 complex, has been extensively studied, little is known about their physiological roles in the producing organisms (reviewed in Ref.…”

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TheStreptomycesSubtilisin Inhibitor (SSI) Gene inStreptomyces coelicolorA3(2)

Kato

Hirano

Ohnishi

et al. 2005

Bioscience, Biotechnology, and Biochemistry

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Comparative studies on the primary structures and inhibitory properties of subtilisin‐trypsin inhibitors from Streptomyces

Cited by 26 publications

References 27 publications

Three Chymotrypsin Genes Are Members of the AdpA Regulon in the A-Factor Regulatory Cascade in Streptomyces griseus

Three Chymotrypsin Genes Are Members of the AdpA Regulon in the A-Factor Regulatory Cascade in Streptomyces griseus

Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor

TheStreptomycesSubtilisin Inhibitor (SSI) Gene inStreptomyces coelicolorA3(2)

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