RESUMO
Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-beta-naphthylamide (APA-beta NH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN' in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-beta NH-Nap substrate compared to their non-deleted parental strains.
Assuntos
Endopeptidases/genética , Genes Bacterianos , Streptomyces/enzimologia , Streptomyces/genética , Subtilisinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Endopeptidases/metabolismo , Deleção de Genes , Biblioteca Genômica , Dados de Sequência Molecular , Oligopeptídeos/química , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato , Subtilisinas/metabolismoRESUMO
A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.
Assuntos
Proteínas de Bactérias/genética , Endopeptidases/genética , Genes Bacterianos , Lipoproteínas/genética , Streptomyces/genética , Sequência de Aminoácidos , Aminopeptidases , Sequência de Bases , Parede Celular/enzimologia , Cromossomos Bacterianos/genética , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases/metabolismo , Escherichia coli/genética , Deleção de Genes , Biblioteca Genômica , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.
Assuntos
Endopeptidases/genética , Genes Bacterianos , Streptomyces/enzimologia , Streptomyces/genética , Sequência de Aminoácidos , Aminopeptidases , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Dipeptidil Peptidases e Tripeptidil Peptidases , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , Sinais Direcionadores de Proteínas/genética , Mapeamento por Restrição , Especificidade da EspécieRESUMO
The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-beta-naphthylamide or arginine-beta-naphthylamide in a liquid overlayer on colonies growing on agar medium to screen for overproduction of the ability to hydrolyse the substrates. The nucleotide sequence of pepN was determined and shown to encode a 95-kDa protein, which displayed significant homology to PepN proteins from other organisms. Analysis of the overproduced proteinase confirmed that this protein was located intracellularly as a monomeric active species. PepN is a metallo-exopeptidase cleaving next to Leu, Arg and Lys in peptide-bond-containing substrates.
Assuntos
Aminopeptidases/genética , Genes Bacterianos/genética , Streptomyces/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/metabolismo , Arginina/análogos & derivados , Proteínas de Bactérias/análise , Sequência de Bases , Antígenos CD13 , Clonagem Molecular , Meios de Cultura , Biblioteca Genômica , Leucina/análogos & derivados , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
We have investigated the aminopeptidase activities present in Streptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.
