High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.     

A novel member of the subtilisin-like protease family from Streptomyces albogriseolus.

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.     

Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.     

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'.     

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces

Summary Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.Abbreviations SD Shine-Dalgarno - SSI Streptomyces subtilisin inhibitor     

Interactions of Streptomyces subtilisin inhibitor with Streptomyces griseus proteases A and B. Enzyme kinetic and computer simulation studies

Streptomyces subtilisin inhibitor (SSI), a dimeric protein that strongly inhibits subtilisins, was shown to form tight inhibitory complexes with Streptomyces griseus proteases A and B (SGPA and SGPB). The apparent dissociation constants of the SGPA-SSI and SGPB-SSI complexes were found to be orders of magnitude less than those of subtilisin-SSI complexes. Using the known atomic coordinates for SGPA and SSI, the highly complementary nature of the surface geometries of the two proteins was confirmed by a computer graphics study, which led to a proposed structure for the SGPA-SSI complex. Kinetic studies further suggested that the SSI dimer can bind two molecules of either SGPA or SGPB, and the 2:1-complexes (consisting of one inhibitor dimer and one enzyme molecule) apparently possess lower intrinsic dissociation constants than the 2:2-complexes. It was also shown that both of SGPA and SGPB are inhibited by both soybean trypsin inhibitor (Kunitz) and bovine pancreatic trypsin inhibitor (Kunitz), but far less strongly than by SSI.     

A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.     

Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20).

The serine proteinase gene (sep) in Aspergillus flavus was disrupted by homologous recombination with a hygromycin resistance gene as the marker. The gene-disrupted mutant GR-2 contained a single-copy insertion of the marker gene and did not express the sep gene. Serine proteinase activity, 36-kDa protein labeled by 3H-diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the culture fluid of GR-2. Despite the absence of the serine proteinase, the total elastinolytic activity levels in the mutant and the wild-type A.flavus were comparable. Immunoblots revealed that the mutant secreted greater amounts of an elastinolytic metalloproteinase gene (mep20) product than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in the wild type. Inhibition of the serine proteinase by Streptomyces subtilisin inhibitor (SSI) in the culture medium of wild-type A.flavus also resulted in an elevation of mep20 gene products. Although no serine proteinase activity could be detected, the level of elastinolytic activity of the SSI-treated culture was comparable to that of the control. Immunoblots revealed that the addition of SSI caused an elevation in the levels of metalloproteinase and its mRNA. These results suggest that the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mechanism to sense the status of extracellular proteolytic activities.     

Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5.

We recently described the isolation and sequence analysis of the daunomycin polyketide synthase biosynthesis genes of Streptomyces sp. strain C5 (J. Ye, M. L. Dickens, R. Plater, Y. Li, J. Lawrence, and W. R. Strohl, J. Bacteriol. 176:6270-6280, 1994). Contiguous to the daunomycin polyketide synthase biosynthesis gene region in Streptomyces sp. strain C5 are four additional genes involved in daunomycin biosynthesis, two of the products of which show similarity to different types of methyltransferases. The dauC gene, encoding aklanonic acid methyltransferase (AAMT), complements dauC-blocked mutants of Streptomyces sp. strain C5, restores in vitro AAMT activities to the mutant strains, and confers in vitro AAMT activity on Streptomyces lividans. Partial purification through gel filtration, followed by photoaffinity labeling of enriched AAMT with S-adenosyl-L-[3H-methyl]methionine, indicates that AAMT is a homodimer with an M(r) of ca. 48,000 (subunit M(r) of ca. 24,000), which corresponds with the size of the deduced gene product. The dauD gene, encoding aklanonic acid methyl ester cyclase, is divergently arranged with respect to dauC. Immediately downstream and apparently translationally coupled with dauD is the dauK gene, encoding carminomycin 4-O-methyltransferase. The dauK gene confers in vitro carminomycin 4-O-methyltransferase activity on S. lividans and is nearly identical to a similar gene isolated from Streptomyces peucetius and characterized. Directly downstream of dauK lies a gene encoding a deduced protein that is similar to the methyl esterases.     

Complex of subtilisin BPN' with Streptomyces subtilisin inhibitor. Complex formation concomitant with change in reducibility of disulfide bonds in the inhibitor

Structure of the complex of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' was studied by examining the thermal denaturation and reducibility of disulfide bonds. The denaturation temperature of the complex was significantly higher than that of the enzyme. Two disulfide bonds localized in the inhibitor side were completely reduced in the complex, whereas only one of them was reduced in the free SSI. Gel filtration of the reduced complex solution showed clearly that the main products of reduction of the complex were two peptide fragments of SSI divided at the active site. The resistive disulfide bond in the complexed inhibitor became accessible as a result of a large conformational change due to splitting of the half-reduced inhibitor.     

Genetic architecture of the polyketide synthases for methymycin and pikromycin series macrolides

The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.     

Gene controlled by promoter--PTH4 depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--P_TH4 which is related to Streptomyces differentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens.

The Streptomyces mycarofaciens mdmB gene encodes a 3-O-acyltransferase that catalyzes the addition of acetyl and propionyl groups to position 3 of the lactone ring in 16-member macrolide antibiotics like midecamycin and spiramycin. A putative O-methyltransferase gene (mdmC) is immediately downstream of mdmB, and both of these genes are closely linked to the mdmA midecamycin resistance gene.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Gene controlled by promoter--P_(TH4) depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.