Partial Amino Acid Sequence of an Alkaline Protease Inhibitor, API-2 (b and c)

Suzuki, Keitarou; Uyeda, Masaru; Shibata, Motoo

doi:10.1080/00021369.1981.10864572

Cited by 6 publications

(5 citation statements)

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“…It has been reported that bacterial serine protease inhibitors from Streptomyces spp. (25,27,29,30), B. subtilis (18), and E. coli (5) inhibit various serine proteases such as trypsin, chymotrypsin, and subtilisin and that a metalloprotease inhibitor of S. nigrescens (20) inhibits thermolysin and other bacterial metalloproteases. In terms of structure-function relationships of proteins, SmaPI would be a good model protein for investigating specific inhibition mechanisms between inhibitor proteins and protease and will contribute to design and development of protease inhibitors exhibiting a high specificity for a given protease.…”

Section: Discussionmentioning

confidence: 99%

“…Subtilisin inhibitor (SSI) of Streptomyces albogriseolus S-3253 was the first proteinaceous protease inhibitor to be isolated from bacteria (25). Subsequently, three similar serine protease inhibitors, such as plasminostreptin from Streptomyces antifibrinolyticus (27), API-2cЈ from Streptomyces griseoincarnatus (29), and SLPI from Streptomyces lividans (30) were isolated. This SSI family has been relatively well characterized in terms of their structure-function relationships (17,19).…”

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confidence: 99%

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Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens

Kim

et al. 1995

Appl Environ Microbiol

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ATCC 27117 produced very small amounts (0.8 U ml ؊1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25؇C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml ؊1 that is also localized in the periplasm of cells at 25؇C. However, a large amount of SmaPI (86 U ml ؊1) was extracellularly produced at the supraoptimal growth temperature of 37؇C from the recombinant S. marcescens(pSP2). We purified SmaPI from the culture supernatant of S. marcescens(pSP2) grown at 37؇C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens

Kim

et al. 1995

Appl Environ Microbiol

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“…Finding that homologous proteins of SSI are widely distributed in streptomycetes, particularly in two genera, Streptomyces and Streptoverticillium (traditional classification), we have recently designated them ''SSIlike proteins'' (Taguchi et al 1992(Taguchi et al , 1993a, here abbreviated to ''SIL'' proteins for those in Streptomyces and ''SIL-V'' proteins for those in Streptoverticillium. To date, plasminostreptin (PSN) from Streptomyces antifibrinolyticus (Sugino et al 1978), alkaline protease inhibitor (API-2cЈ) from Streptomyces griseoincarnatus (Suzuki et al 1981), Streptomyces lividans protease inhibitor (SLPI) or Streptomyces trypsin inhibitor 1 (STI1) from Streptomyces lividans (Ueda et al 1992;Strickler et al 1992), STI2 from Streptomyces longisporus (Strickler et al 1992), and SAC-I from Streptoverticillium cinnamoneum (Tanabe et al 1994) have been isolated independently by us and other groups as SSI-like proteins, and their structure-function relationships have been well characterized Taguchi et al 1994;Terabe et al 1994aTerabe et al ,b, 1995Terabe et al , 1996Ueda et al 1992). To our knowledge, no other bacterial protease inhibitor family has been found.…”

Section: Introductionmentioning

confidence: 99%

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

et al. 1997

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We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them "SSI-like proteins" (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.

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“…Sequence alignment and dendrogram of kexstatin I and other SSI-family inhibitors(A) Alignment of amino acid sequences of the mature forms of native kexstatin I (shown in bold) and other SSI-family inhibitors : SSI[20,21] ; SCI, Streptomyces chymotrypsin inhibitor ; PSN, plasminostrepin[28] ; STIs 1 and 2, Streptomyces trypsin inhibitors 1 and 2[29] ; API, alkaline proteinase inhibitor 2c[30] ; and SILs 1, 2, 3, 4, 8, 10, 13 and 14, Streptomyces subtilisin inhibitorlike proteins 1, 2, 3, 4, 8, 10, 13 and 14[31]. Identical amino acid residues among all of the inhibitors listed are shadowed.…”

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Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase

Oda

OYAMA

Ito

et al. 2001

Biochemical Journal

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Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr69-Lys70↓Glu71-, where ↓ indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P1 and P2 sites, conversion of the Thr69-Lys70 segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr69-Lys70 sequence was replaced by the Thr69-Arg70, Lys69-Lys70 and Lys69-Arg70 sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys69-Arg70 mutant was the most potent inhibitor. The Ki for Kex2 proteinase was 3.2×10-10 M, which was 140-fold lower than that of the inhibitor with the Thr69-Lys70 sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

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Partial Amino Acid Sequence of an Alkaline Protease Inhibitor, API-2 (b and c)

Cited by 6 publications

References 10 publications

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase

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