The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66

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

Gene

Volume 141, Issue 1, 8 April 1994, Pages 115-119

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Abstract

The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-β-naphthylamide or arginine-β-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 intracellulariy as a monomeric active species. PepN is a metallo-exopeptidase cleaving next to Leu, Arg and Lys in peptide-bond-containing substrates.

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Cited by (19)

Metabolic perturbations of Lolium perenne L. by enrofloxacin: Bioaccumulation and multistage defense system
2022, Journal of Hazardous Materials
Citation Excerpt :
It is reported that the molecular complex formed by GSH and organic matter can be combined with glutathione hydrolase to generate more easily degraded byproducts (Kurade et al., 2021). Simultaneously, the significantly increased expression of cys-gly metallodipeptidase (DUG1) and aminopeptidase N (pepN) also promoted the metabolism of GSH (Kaur et al., 2009; Butler et al., 1994). The glutathione reductase encoded by GSR converts glutathione disulfide (GSSG) into GSH.
Plants are readily exposed to the antibiotics residues in reclaimed water indicating an urgent need to comprehensively analyze their ecotoxicological effects and fate of these emerging contaminants. Here, we unraveled the dissemination of enrofloxacin (ENR) in a pasture grass, Lolium perenne L., and identified multistage defense systems as its adaptation mechanism. Uptaken concentrations of ENR ranged from 1.28 to 246.60 µg g⁻¹ with bioconcentration factors (BCF) upto 15.13, and translocation factors (TF) upto 0.332. The antioxidant enzymatic activities such as superoxide dismutase, peroxidase, and catalase were increased by upto 115%. Further transcriptomics demonstrated that differentially expressed genes (DEGs) involved in glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and glutathione metabolism were significantly up-regulated by 1.56–5.93, 3–7 and 1.04–6.42 times, respectively; whilst, the DEGs in nitrogen and sulfur metabolism pathways were significantly up-regulated by 1.06–5.64 and 2.64–3.54 folds. These processes can supply energy, signaling molecules, and antioxidants for detoxification of ENR in ryegrass. Such results provide understanding into fasting grass adaptability to antibiotics by enhancing the key protective pathways under organic pollutant stresses in environments.
Aminopeptidase N (Streptomyces lividans)
2013, Handbook of Proteolytic Enzymes
pTONA5: A hyperexpression vector in streptomycetes
2008, Protein Expression and Purification
We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases—leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)—using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth.
Aminopeptidase N (Streptomyces lividans)
2004, Handbook of Proteolytic Enzymes, Second Edition: Volume 1: Aspartic and Metallo Peptidases
This chapter reviews the activity, specificity and structural chemistry of Streptomyces lividans aminopeptidase N protein. The gene encoding the S. lividans aminopeptidase N protein was isolated from a screening experiment to characterize the major aminopeptidase activities in the strain. Nucleotide sequence determination of the cloned genomic DNA fragments revealed a predicted protein of approximately 95 kDa. The comparison of the predicted protein sequence with databases displayed a strong similarity to aminopeptidase N proteins from several species, with that of the gram-positive bacterium Lactococcus lactis being the closest match. The PepN was strongly overexpressed by strains carrying the gene on a multicopy plasmid. No secretion signal sequence was visible at the N-terminus of the predicted protein, suggesting that the PepN is primarily located intracellularly. It is found that after 40 hours of fermentation, significant amounts of activity could be detected in the culture medium. Analysis showed that the PepN protein appeared to be relatively enriched compared to the other intracellular proteins and therefore, PepN was purified from the medium.
Bacterial aminopeptidases: Properties and functions
1996, FEMS Microbiology Reviews
Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasec activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty genes encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine aminopeptidase from Escherichia coli and the leucine aminopeptidase from Aeromonas proteolytica, have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their substrate specificity: broad or narrow. These enzymes can also be classified by another criterium based on their catalytic mechanism: metallo-, cysteine- and serine-aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised peptide chains (methionine aminopeptidases), the stabilisation of multicopy ColE1 based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pcp) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.
Advances in heterologous gene expression by Streptomyces
1994, Current Opinion in Biotechnology
During the past year, noteworthy advances have been made in our ability to express foreign genes in Streptomyces. These advances were due, in part, to a detailed examination of the critical parameters that limit expression by Streptomyces of soluble forms of the human T-cell receptor CD4. Significant progress has also been made in our understanding of transcriptional regulation and protease gene expression. Application of this knowledge to expression vector design and the construction of alternative expression hosts should improve our ability to easily and routinely express foreign genes in Streptomyces.

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Abstract

References (22)

Basic local alignment search tool

J. Mol. Biol.

Secretory synthesis of human interleukin-2 by Streptomyces lividans

Gene

Cloning and characterization of an aminopeptidase P-encoding gene from Streptomyces lividans

Gene

Two novel Streptomyces protein protease inhibitors

J. Biol. Chem.

Improved M 13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Gene

Purification and properties of an extracellular aminopeptidase from Streptomyces lividans 1326

J. Gen. Microbiol.

Proteolytic enzymes from recombinant Streptomyces lividans TK24

FEMS Microbiol. Lett.

Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution

Eur. J. Biochem.

Expression of the soluble CD4 receptor in Streptomyces

J. Cell Biochem. Suppl.

Cloning of genetic loci involved in endoprotease activity in S. livi-dans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene

Can. J. Microbiol.

Personal access of sequence databases on personal computers

Nucleic Acids Res.

Cited by (19)

Metabolic perturbations of Lolium perenne L. by enrofloxacin: Bioaccumulation and multistage defense system

Aminopeptidase N (Streptomyces lividans)

pTONA5: A hyperexpression vector in streptomycetes

Aminopeptidase N (Streptomyces lividans)

Bacterial aminopeptidases: Properties and functions

Advances in heterologous gene expression by Streptomyces