The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66

Butler, Michael J.; Aphale, Jayant S.; Binnie, Craig; DiZonno, M A; Krygsman, Phyllis; Soltes, Glenn; Walczyk, Eva; Malek, Lawrence T.

doi:10.1016/0378-1119(94)90137-6

Cited by 18 publications

(5 citation statements)

References 17 publications

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“…Several monopeptidyl, dipeptidyl and tripeptidyl aminopeptidases of Streptomyces spp. have been identified, none with any proteolytic activity against chromophoric tetrapeptides [17–24]. Moreover, the better characterized tripeptidyl aminopeptidase (TAP) from Streptomyces lividans 66 obviously has inappropriate specificity (Ala‐Pro‐Ala↓naphthylamide) for performing the final TGase processing [17, 20].…”

mentioning

confidence: 99%

Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step

Zotzel¹,

Pasternack²,

Pelzer³

et al. 2003

European Journal of Biochemistry

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Transglutaminase (TGase) from Streptomyces mobaraensis is secreted as a precursor protein which is completely activated by the endoprotease TAMEP, a member of the M4 protease family [Zotzel, J., Keller, P. & Fuchsbauer, H.‐L. (2003) Eur. J. Biochem. 270, 3214–3222]. In contrast with the mature enzyme, TAMEP‐activated TGase exhibits an additional N‐terminal tetrapeptide (Phe‐Arg‐Ala‐Pro) suggesting truncation, at least, by a second protease. We have now isolated from the culture broth of submerged colonies a tripeptidyl aminopeptidase (SM‐TAP) that is able to remove the remaining tetrapeptide. The 53‐kDa peptidase was purified by ion‐exchange and phenyl‐Sepharose chromatography and subsequently characterized. Its proteolytic activity was highest against chromophoric tripeptides at pH 7 in the presence of 2 mm CaCl2. EDTA and EGTA (10 mm) both diminished the proteolytic activity by half. Complete inhibition was only achieved with 1 mm phenylmethanesulfonyl fluoride, suggesting that SM‐TAP is a serine protease. Alignment of the N‐terminal sequence confirmed its close relation to the Streptomyces TAPs. That removal of Phe‐Arg‐Ala‐Pro from TAMEP‐activated TGase by SM‐TAP occurs in a single step was confirmed by experiments using various TGase fragments and synthetic peptides. SM‐TAP was also capable of generating the mature N‐terminus by cleavage of RAP‐TGase. However, AP‐TGase remained unchanged. As SM‐TAP activity against chromophoric amino acids such as Pro‐pNA or Phe‐pNA could not be detected, the tetrapeptide of TAMEP‐activated TGase must be removed without formation of an intermediate.

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mentioning

confidence: 99%

Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step

Zotzel¹,

Pasternack²,

Pelzer³

et al. 2003

European Journal of Biochemistry

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“…In S. lividans, similar secretory bottlenecks may exist when heterologous proteins are expressed, although our knowledge about this is quite fragmentary. The endogenous proteases produced by S. lividans intracellularly (Butler et al, 1994), extracellularly (Lichenstein et al, 1992;Butler et al, 1995;Krieger et al, 1994;Mori & Ito, 2001) or cell wall-associated (Binnie et al, 1995) have been investigated to be bottlenecks for the production of some heterologous proteins. The results obtained here show that the 20S proteasome seems to be a bottleneck for some heterologous proteins produced in S. lividans such as shuTNFRII and sCT but not for others as illustrated with shuTNFRI.…”

Section: Discussionmentioning

confidence: 99%

“…From then on, degradation of the heterologous proteins by endogenous protease activity of S. lividans has been well-documented, and intracellular and extracellular proteases and proteases associated with the mycelium have been described (e.g. Binnie et al, 1995;Butler et al, 1994;Krieger et al, 1994). Recently, the 20S proteasome, a self-compartmentalizing proteolytic system, was identified in Streptomyces (De Mot et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins

et al. 2005

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Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.

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“…Each fraction contained a mixture of three amino acid residues that coincided with the degradation of a nested set of esterase species with different N termini, consisting of 25% of the recombinant esterase secreted by S. lividans(pCT1), 50% of the mature protein lacking the N-terminal alanine residue, and 25% which lacked two alanine residues. Obviously, a proteolytic processing at the amino terminus of the esterase occurred during incubation with [1, H]DFP catalyzed by peptidases produced by S. lividans (2,13). In each case, [1, H]DFP labeling detected in fractions 10 to 12 was specific to Ser-11, indicating that this residue in the sequence GDSYT might be the active serine.…”

Section: Analysis Of the Esta Gene And The Deduced Proteinmentioning

confidence: 98%

Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes

et al. 1996

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An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 ؎ 5.8 Da, as determined by electrospray mass spectrometry. The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S. diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702. Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35-or 36-residue signal peptide for secretion in S. lividans or S. diastatochromogenes, respectively. The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S. diastatochromogenes. The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies. Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes. A serine modified by [1,3-3 H]diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT. This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.Streptomycetes are gram-positive, saprophytic soil microorganisms that use a wide variety of extracellular hydrolytic enzymes, including chitinases, cellulases, xylanases, proteases, lipases, and nucleases (50), to degrade organic material in the soil. Polysaccharidases, proteases, and enzymes exhibiting unusual catalytic activities have been studied extensively (35). However, except for phospholipase D, only a few streptomycete lipolytic enzymes and their corresponding genes have been analyzed so far, although Sztajer et al. (46) reported high lipolytic activity in Streptomyces strains. An esterase secreted by the plant-pathogenic strains of Streptomyces scabies has been characterized at the protein (31) and DNA (40) levels. The enzyme is believed to be important in pathogenicity and penetration of S. scabies by hydrolyzing ester bonds in suberin, a waxy polyester covering the external portions of the plants. Recently, genes encoding extracellular lipases from Streptomyces sp. strain M11 (36) and Streptomyces albus G (17) have been cloned and sequenced.Esterases and lipases are carboxylic ester hydrolases (EC 3.1.1). The carboxylesterases (EC 3.1.1.1) hydrolyze water-soluble or emulsified esters with relatively short fatty acid chains, whereas lipases (triacylglycerol acyl hydrolases; EC 3.1.1.3) preferentially act on emulsified substrates with long-chain fatty acids. The hydrolytic mechanism of most of the known esterases and lipases resembles that of serine proteases. These hydrolases all contain a similar catalytic triad, generally consisting of a nucleophilic serine residue that acts in conjunction with a histidine and an aspartic acid residue (8,10,18,51). Microbial lipolytic enzymes have become biotechnologically important enzymes used for hydrol...

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The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66

Cited by 18 publications

References 17 publications

Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step

Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step

Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins

Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes

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