Studies on alkaline protease inhibitor (API-2) produced by Streptomyces griseoincarnatus strain No. KTo-250. Part VII. Partial amino acid sequence of an alkaline protease inhibitor, API-2 (b and c).

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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Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

Taguchi

Kojima

Terabe

et al. 1997

J Mol Evol

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H n.m.r. spectrum from the flexible N‐terminal segment of Streptomyces subtilisin inhibitor

Akasaka

1985

International Journal of Peptide and Protein Research

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The 'H n.m.r. spectrum of Streptomyces subtilisin inhibitor shows a limited number of unusually sharp signals at room temperature. Some of these signals are assigned uniquely to protons of the side chains of the N-terminal segment, Aspl-Ala2-Pro3-Ser4-AlaS-Leu6-Tyr7-based on experiments of spin decoupling, pH titration, and enzymatic cleavage of the protein. Quantitative examination of these signals indicates that the N-terminal end of this protein is heterogeneous in that the protein contains a considerable fraction whose sequence starts with Ala2 rather than with Aspl. The pK, values for the amino groups of Aspl and Ala2 exposed at the N-terminus are determined to be 8.9 * 0.4 and 9.0 * 0.1, respectively. Furthermore, examination of the line-widths of the methyl proton resonances of Ala2 and Alas residues indicates that the N-terminal peptide segment is free and undergoes rapid segmental motions in the order of lo-' s.

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

Taguchi

Kojima

Terabe

et al. 1994

European Journal of Biochemistry

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Three novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor-like (SIL) inhibitors were isolated from culture supernatant of Streptomyces ; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SILA from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg-Glu for SIL2 and SIL3, and Lys-Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor-subtilisin complex under acidic conditions. The presence of an argininefiysine residue at the P1 site was in agreement with their trypsin-inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in P-sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (K,) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.Proteinaceous protease inhibitors, which play an important role in inhibiting the proteolytic activity of proteases, are widely distributed in animals, plants and microorganisms [ 11, and have a variety of structures and protease specificities. Among them, inhibitors of serine proteases have been well studied, and are divided into several groups according to their structures and the locations of disulfide bridges [ 2 ] . Streptomyces subtilisin inhibitor (SSI), discovered from culture broth of Streptomyces albogriseolus S-3253, is the best characterized protein [3] [7]. Recently, in our study, we have found that SSI-like (SIL) protease inhibitors are distributed at high frequency among streptomycetes [8 -101, although their physiological role in Correspondence to S . Taguchi,

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Studies on alkaline protease inhibitor (API-2) produced by Streptomyces griseoincarnatus strain No. KTo-250. Part VII. Partial amino acid sequence of an alkaline protease inhibitor, API-2 (b and c).

Cited by 6 publications

References 3 publications

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

H n.m.r. spectrum from the flexible N‐terminal segment of Streptomyces subtilisin inhibitor

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

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