小島 修一

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 mu g/ml of SGT did not exceed 15%, This low yield was assumed to be due mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refolding media were examined for their ability to minimize misfolding. As a result, 1 M Tris, 1 M diethanolamine, and 1 M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding, The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-terminal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr. Of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2ml, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease. (C) 1999 Academic Press.

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 mu g/ml of SGT did not exceed 15%, This low yield was assumed to be due mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refolding media were examined for their ability to minimize misfolding. As a result, 1 M Tris, 1 M diethanolamine, and 1 M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding, The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.

Aqualysin I is an alkaline serine protease isolated from Thermus aquaticus YT-1, an extreme thermophile. We have measured the P1-specificity of aqualysin I, using wildtype and five P1-substituted derivatives of Streptomyces subtilisin inhibitor (SSI). SSIs efficiently inhibited the activity of aqualysin I, with low substrate specificity. Charge and hydrophobicity of side chain of the P1 amino acid residue showed no significant effect to the P1-specificity of this enzyme.

We have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J, Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-PEG, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253, Complete amino acid sequence determination revealed that SAM-PEG is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-PEG, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-PBO. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-PEG estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Pa) and that of the S-pyridylethylated form (18,808.4 Dal of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SARI-PEG exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-PEG exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degrees C. The complex formation of SAM-PEG with SSI was confirmed by native-PAGE analysis.

Aqualysin I is an alkaline serine protease isolated from Thermus aquaticus YT-1, an extreme thermophile. We have measured the P1-specificity of aqualysin I, using wildtype and five P1-substituted derivatives of Streptomyces subtilisin inhibitor (SSI). SSIs efficiently inhibited the activity of aqualysin I, with low substrate specificity. Charge and hydrophobicity of side chain of the P1 amino acid residue showed no significant effect to the P1-specificity of this enzyme.

We have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J, Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-PEG, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253, Complete amino acid sequence determination revealed that SAM-PEG is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-PEG, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-PBO. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-PEG estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Pa) and that of the S-pyridylethylated form (18,808.4 Dal of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SARI-PEG exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-PEG exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degrees C. The complex formation of SAM-PEG with SSI was confirmed by native-PAGE analysis.

Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous "serine protease inhibitors" produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a ''His-Glu-Xaa-Xaa-His'' motif of SGMPII was identified as the residue modified by CICH2CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.

Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous "serine protease inhibitors" produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a ''His-Glu-Xaa-Xaa-His'' motif of SGMPII was identified as the residue modified by CICH2CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli, A 1.6-kbp DNA fragment, isolated from D, vulgaris (Miyazaki F) by double digestion with Sail and EcoRI, contained the flavodoxin gene and its regulatory region, An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E, coli, The purified protein was soluble and exhibited a characteristic visible absorption spectrum, HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D, vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM, In vitro H-2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E-1 = -434 and E-2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed time feature of a conserved Glu residue at the surface of the molecule.

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli, A 1.6-kbp DNA fragment, isolated from D, vulgaris (Miyazaki F) by double digestion with Sail and EcoRI, contained the flavodoxin gene and its regulatory region, An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E, coli, The purified protein was soluble and exhibited a characteristic visible absorption spectrum, HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D, vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM, In vitro H-2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E-1 = -434 and E-2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed time feature of a conserved Glu residue at the surface of the molecule.

The propeptide of subtilisin BPN', located between a signal peptide and the mature region of the protease, is known to exhibit inhibitory activity toward subtilisin BPN', in addition to its activity as an intramolecular chaperone that facilitates folding of subtilisin BPN'. Another unique feature is that although the isolated propeptide is in a random-coil state, it forms a defined tertiary structure when it is bound to subtilisin BPN'. In this study, amino acid replacements likely to increase the hydrophobicity of the propeptide have been introduced so that the isolated propeptide forms a defined tertiary structure. By successive replacements of Ala47 by Phe, Gly13 by lie and Val65 by lie, the propeptide was found to form a tertiary structure in addition to an increase in its secondary structure content, which were identified by circular dichoism spectra measurements. Concurrently, the propeptide, which is a temporary inhibitor in its wild-type form, became resistant to proteolytic digestion by subtilisin BPN'. These results show not only the close relationship between tertiary structure formation in the propeptide and its function as a protease inhibitor but also the ability of a random-coil protein to form a tertiary structure after a limited number of well-designed amino acid replacements. (C) 1998 Academic Press Limited.

The propeptide of subtilisin BPN', located between a signal peptide and the mature region of the protease, is known to exhibit inhibitory activity toward subtilisin BPN', in addition to its activity as an intramolecular chaperone that facilitates folding of subtilisin BPN'. Another unique feature is that although the isolated propeptide is in a random-coil state, it forms a defined tertiary structure when it is bound to subtilisin BPN'. In this study, amino acid replacements likely to increase the hydrophobicity of the propeptide have been introduced so that the isolated propeptide forms a defined tertiary structure. By successive replacements of Ala47 by Phe, Gly13 by lie and Val65 by lie, the propeptide was found to form a tertiary structure in addition to an increase in its secondary structure content, which were identified by circular dichoism spectra measurements. Concurrently, the propeptide, which is a temporary inhibitor in its wild-type form, became resistant to proteolytic digestion by subtilisin BPN'. These results show not only the close relationship between tertiary structure formation in the propeptide and its function as a protease inhibitor but also the ability of a random-coil protein to form a tertiary structure after a limited number of well-designed amino acid replacements. (C) 1998 Academic Press Limited.

The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone, For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli, Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates, The main cleavage site was identified as Glu(53)-Lys(54), with minor sites at Thr(17)-Met(18) and Met(21)-Ser(22), which were located in turn regions of the propeptide in the complex with subtilisin BPN'. Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin. Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor, (C) 1997 Federation of European Biochemical Societies.

The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone, For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli, Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates, The main cleavage site was identified as Glu(53)-Lys(54), with minor sites at Thr(17)-Met(18) and Met(21)-Ser(22), which were located in turn regions of the propeptide in the complex with subtilisin BPN'. Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin. Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor, (C) 1997 Federation of European Biochemical Societies.

The eukaryotic initiation factor eIF-4E from Xenopus laevis was expressed in Escherichia coli and refolded in an active form. To define the cysteine residues forming a disulfide bond in Xenopus eIF-4E, each of the 3 cysteine residues was changed to serine by site-directed mutagenesis. Cap-binding activities of the mutant proteins were evaluated by 7-methyl-GTP(m7GTP)-affinity column chromatography. Even the mutant protein containing no cysteine showed an affinity for m7GTP. From the above results and the estimation of the sulfhydryl groups by Ellman's assay method, we concluded that a disulfide bond is not involved in the active Xenopus eIF-4E.

The eukaryotic initiation factor eIF-4E from Xenopus laevis was expressed in Escherichia coli and refolded in an active form. To define the cysteine residues forming a disulfide bond in Xenopus eIF-4E, each of the 3 cysteine residues was changed to serine by site-directed mutagenesis. Cap-binding activities of the mutant proteins were evaluated by 7-methyl-GTP(m(7) GTP)-affinity column chromatography. Even the mutant protein containing no cysteine showed an affinity for m(7)GTP. From the above results and the estimation of the sulfhydryl groups by Ellman's assay method, we concluded that a disulfide bond is not involved in the active Xenopus elF-4E. (C) 1997 Federation of European Biochemical Societies.

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 Pi 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.

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 Pi 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.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki Fl was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene(rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that an responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D, vulgaris rubredoxin. in vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its odor potential was determined as -5 mV.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki Fl was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene(rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that an responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D, vulgaris rubredoxin. in vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its odor potential was determined as -5 mV.

We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y.,Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin] BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.

We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y.,Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin] BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.

The alpha(3)-peptide, which comprises three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala, was designed to form an amphipathic alpha-helix with a hydrophobic surface by Leu residues and a hydrophilic surface by Glu and Lys residues, thus yielding a coiled coil or a helical bundle structure through their association. The alpha(3)-peptide was produced by gene engineering using Escherichia coli. Association to a tetramer had been demonstrated by sedimentation equilibrium analysis in a previous study. In addition to tetramerization, electron microscopic observation revealed that the alpha(3)-peptide formed ''fibrils'' 5 to 10 nm in width in 10 mM potassium phosphate/0.1 M KCl (pH 6.0). By increasing the salt concentration, the alpha(3)-peptide formed much larger ''fibers'' assembled from many ''filaments'' running along the long axis, each of which was thinner and longer than those observed at lower salt concentration. Hydrophobic interaction is considered to be a main force responsible for forming the fibrous structure. However, the electrostatic features of the alpha(3)-peptide seem to affect fibril assembly, since the shape and size of the fibrous structure were altered by the ionic strength of the solution. To our knowledge, this is the first report to describe formation of a fibrous structure by a de novo designed alpha-helix-forming peptide, and thus the alpha(3)-peptide with its simple sequence is considered to be a potential model peptide for investigating the molecular mechanisms of fibril formation by peptides or proteins.

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of the squash-type protease inhibitor family, is composed of 29 amino acids and shows strong inhibition of trypsin by its compact structure, To study the structure-function relationship of this inhibitor using protein engineering methods, we constructed an expression system for CMTI-I as a fused protein with porcine adenylate kinase (ADK), A Met residue was introduced into the junction of ADK and CMTI-I to cleave the fusion protein,vith CNBr, whereas a Met at position 8 of authentic CMTI-I was replaced by Leu, Escherichia call JM109 transformed with the constructed plasmid expressed the fused protein as an inclusion body, After cleavage of the expressed protein with CNBr, fully reduced species of CMTI-I were purified by reversed-phase HPLC and then oxidized with air by shaking, For efficient refolding of CMTI-I, we used 50 mM NH4HCO3 (pH 7.8) containing 0.1% PEG 6000 at higher protein concentration, Strong inhibitory activity toward trypsin was detected only in the first of three HPLC peaks, The inhibitor constant of CMTI-I thus obtained, in which Met8 was replaced by Leu, was 1.4x10(-10) M, The effect of replacement of Met with Leu at position 8 was shown to be small by comparison of the inhibitor constant of authentic CMTI-III bearing Lys at position 9 (8.9x10(-11) M) with that of its mutant bearing Leu at position 8 and Lys at position 9 (1.8x10(-10) M), To investigate the role of the well conserved hydrophobic residues of CMTI-I in its interaction with trypsin, CMTI-I mutants in which one or all of the four hydrophobic residues were replaced by Ala were prepared, The inhibitor constants of these mutants indicated that those with single replacements were 5-40 times less effective as trypsin inhibitors and that the quadruple mutant was similar to 450 times less effective, suggesting that the hydrophobic residues in CMTI-I contribute to its tight binding with trypsin, However, each mutant was not converted to a temporary inhibitor.

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of the squash-type protease inhibitor family, is composed of 29 amino acids and shows strong inhibition of trypsin by its compact structure, To study the structure-function relationship of this inhibitor using protein engineering methods, we constructed an expression system for CMTI-I as a fused protein with porcine adenylate kinase (ADK), A Met residue was introduced into the junction of ADK and CMTI-I to cleave the fusion protein,vith CNBr, whereas a Met at position 8 of authentic CMTI-I was replaced by Leu, Escherichia call JM109 transformed with the constructed plasmid expressed the fused protein as an inclusion body, After cleavage of the expressed protein with CNBr, fully reduced species of CMTI-I were purified by reversed-phase HPLC and then oxidized with air by shaking, For efficient refolding of CMTI-I, we used 50 mM NH4HCO3 (pH 7.8) containing 0.1% PEG 6000 at higher protein concentration, Strong inhibitory activity toward trypsin was detected only in the first of three HPLC peaks, The inhibitor constant of CMTI-I thus obtained, in which Met8 was replaced by Leu, was 1.4x10(-10) M, The effect of replacement of Met with Leu at position 8 was shown to be small by comparison of the inhibitor constant of authentic CMTI-III bearing Lys at position 9 (8.9x10(-11) M) with that of its mutant bearing Leu at position 8 and Lys at position 9 (1.8x10(-10) M), To investigate the role of the well conserved hydrophobic residues of CMTI-I in its interaction with trypsin, CMTI-I mutants in which one or all of the four hydrophobic residues were replaced by Ala were prepared, The inhibitor constants of these mutants indicated that those with single replacements were 5-40 times less effective as trypsin inhibitors and that the quadruple mutant was similar to 450 times less effective, suggesting that the hydrophobic residues in CMTI-I contribute to its tight binding with trypsin, However, each mutant was not converted to a temporary inhibitor.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was 'alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was 'alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.

Amino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Muller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Muller, but belongs to the same taxon as S. lividans 66.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10) s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8) s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8) s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (>10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10) s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.

Amino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Muller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Muller, but belongs to the same taxon as S. lividans 66.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10) s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8) s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8) s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (>10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10) s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.

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-PZ0, This reactive site is the same as that toward an exogenous target enzyme, subtilisin BPN'.

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S-pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-138). The latter half includes a highly hydrophobic segment, a four-times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy-terminal segment containing Trp126. Ultraviolet absorption and CD spectra of TP4-rat-liver-nucleosome-core-DNA (double-stranded DNA) complexes suggest a TP4-induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trp126) fluorescence of TP4 upon its binding to double-stranded and single-stranded boar liver nucleosome-core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double-stranded and single-stranded DNA were calculated to be 7.3x10(3) M(-1) and 4.1x10(3) M(-1), respectively. These results suggest that TP4, having different domain structures from TP1-3 and a higher affinity for double-stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S-pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-138). The latter half includes a highly hydrophobic segment, a four-times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy-terminal segment containing Trp126. Ultraviolet absorption and CD spectra of TP4-rat-liver-nucleosome-core-DNA (double-stranded DNA) complexes suggest a TP4-induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trp126) fluorescence of TP4 upon its binding to double-stranded and single-stranded boar liver nucleosome-core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double-stranded and single-stranded DNA were calculated to be 7.3x10(3) M(-1) and 4.1x10(3) M(-1), respectively. These results suggest that TP4, having different domain structures from TP1-3 and a higher affinity for double-stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.

We determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family, and then identified its reactive site, SIL15 is composed of 113 amino acids and exists as a dimer, Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gin residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the alpha(1)-helix and the other in the flexible loop region near the reactive site, Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7X10(-11) M) was due to the presence of a Gin residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and turkey ovomucoid domain 3.

A novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin-inhibitor-like (SIL) inhibitor family by sequence analysis of its amino-terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. and Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522-524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward alpha-chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI-family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the beta-sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants K-i obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for alpha-chymotrypsin. The P1 site was identified as methionine, which was in good agreement with the substrate specificity of alpha-chymotrypsin. SSI, which also possesses a methionine residue at the P1 site, inhibits alpha-chymotrypsin poorly (inhibitor constant, 4.0 mu M). Such a difference in the inhibitory properties of SIL8 and SSI toward alpha-chymotrypsin is discussed on the basis of the structures of the inhibitors.

The effect of an engineered disulfide bond between two identical subunits of a dimeric protein, Streptomyces subtilisin inhibitor, on the stability of the protein was studied by differential scanning calorimetry. The introduction of the linkage caused a large stabilization without changing the cooperativity of unfolding, with the denaturation temperature of a 2 mg/mL solution being increased by 14.3 degrees C to 95.0 degrees C at pH 9.5 and by 16.4 degrees C to 63.0 degrees C at pH 3.0. The stabilization was caused by a loss of denaturational entropy, i.e., -40 and -98 cal K-1 mol(-1) at pH 3.0 and 9.5, respectively, which more than compensated for the loss in the denaturational enthalpy.

A novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin-inhibitor-like (SIL) inhibitor family by sequence analysis of its amino-terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. and Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522-524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward alpha-chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI-family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the beta-sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants K-i obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for alpha-chymotrypsin. The P1 site was identified as methionine, which was in good agreement with the substrate specificity of alpha-chymotrypsin. SSI, which also possesses a methionine residue at the P1 site, inhibits alpha-chymotrypsin poorly (inhibitor constant, 4.0 mu M). Such a difference in the inhibitory properties of SIL8 and SSI toward alpha-chymotrypsin is discussed on the basis of the structures of the inhibitors.

The effect of an engineered disulfide bond between two identical subunits of a dimeric protein, Streptomyces subtilisin inhibitor, on the stability of the protein was studied by differential scanning calorimetry. The introduction of the linkage caused a large stabilization without changing the cooperativity of unfolding, with the denaturation temperature of a 2 mg/mL solution being increased by 14.3 degrees C to 95.0 degrees C at pH 9.5 and by 16.4 degrees C to 63.0 degrees C at pH 3.0. The stabilization was caused by a loss of denaturational entropy, i.e., -40 and -98 cal K-1 mol(-1) at pH 3.0 and 9.5, respectively, which more than compensated for the loss in the denaturational enthalpy.

We report here another example of renaturation of subtilisin BPN'(Sbtl) by using an immobilized preparation instead of applying a digestible mutant of Streptomyces subtilisin inhibitor (SSI), a proteinaceous inhibitor of Sbtl [M. Matsubara et al. (1994) FEBS Letters 342, 193-196]. The mature Sbtl was immobilized on agarose beads employing the amino group of the protein. After thorough washing, the immobilized Sbtl was subjected to denaturation in 6 M guanidine hydrochloride (GdnHCl) at pH 2.4 for 4 h, followed by renaturation in 2 M potassium acetate al pH 6.5 for 24 h. This denaturation/renaturation cycle was repeated five times. The recovered activity of the renatured immobilized Sbtl settled at a constant level after the third denaturation/renaturation cycle, demonstrating that almost 100% renaturation was attained by use of the immobilized Sbtl. This immobilized Sbtl preparation could well be utilized for the mechanistic study of protein folding. We then found that 2 M potassium acetate was superior to 2 M potassium chloride as a refolding medium and that the ability of SSI to induce the correct shape of the mature Sbtl was lacking in several refolding media in both thermodynamic and kinetic criteria. Thus the main cause for the increase of refolding yield of Sbtl by coexistence of SSI was prevention of the autolysis of Sbtl.

We report here another example of renaturation of subtilisin BPN'(Sbtl) by using an immobilized preparation instead of applying a digestible mutant of Streptomyces subtilisin inhibitor (SSI), a proteinaceous inhibitor of Sbtl [M. Matsubara et al. (1994) FEBS Letters 342, 193-196]. The mature Sbtl was immobilized on agarose beads employing the amino group of the protein. After thorough washing, the immobilized Sbtl was subjected to denaturation in 6 M guanidine hydrochloride (GdnHCl) at pH 2.4 for 4 h, followed by renaturation in 2 M potassium acetate al pH 6.5 for 24 h. This denaturation/renaturation cycle was repeated five times. The recovered activity of the renatured immobilized Sbtl settled at a constant level after the third denaturation/renaturation cycle, demonstrating that almost 100% renaturation was attained by use of the immobilized Sbtl. This immobilized Sbtl preparation could well be utilized for the mechanistic study of protein folding. We then found that 2 M potassium acetate was superior to 2 M potassium chloride as a refolding medium and that the ability of SSI to induce the correct shape of the mature Sbtl was lacking in several refolding media in both thermodynamic and kinetic criteria. Thus the main cause for the increase of refolding yield of Sbtl by coexistence of SSI was prevention of the autolysis of Sbtl.

Protein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the beta-sheet region. The reactive site of SIL1 was considered to be Arg(70)-Glu(71) from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN' strongly with an inhibitor constant (K-i) of 2.8.10(-11)M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a K-i value of 5.5.10(-8)M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.