Sequencing and High Expression of Aminopeptidase P Gene from Escherichia coli HB101

Yoshimoto, Tadashi; Tone, Hiroshi; Honda, Takashi; Osatomi, Kiyoshi; Kobayashi, Ryûji; Tsuru, Daisuke

doi:10.1093/oxfordjournals.jbchem.a122678

Cited by 46 publications

(28 citation statements)

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“…Protein data base searches using both sequence-and structure-based profiles identified several other proteins likely to share the same fold (18). These proteins included creatinase (19), aminopeptidase P (20,21), proline dipeptidase (22,23), and p67 (24,25), a eukaryotic initiation factor 2 (eIF2)-associated protein proposed to regulate protein synthesis by protecting the eIF2 a subunit from phosphorylation by eIF2 kinases.…”

mentioning

confidence: 99%

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Arfin

Kendall

Hall

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

237

181

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Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a fulllength clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2a and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermusfervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

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mentioning

confidence: 99%

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Arfin

Kendall

Hall

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

237

181

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“…No significant similarities were found between the sequence of pepT and those of E. coli pepN (1,17), pepA (30), and pepP (36) (20); and peptidase D (PepD; amino acids 110 to 152), an E. coli dipeptidase (8).…”

Section: Methodsmentioning

confidence: 99%

Cloning and nucleotide sequence of the anaerobically regulated pepT gene of Salmonella typhimurium

Miller

Bagga

1991

J Bacteriol

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The anaerobically regulated pepT gene of Salmonella typhimurium has been cloned in pBR328. Strains carrying the pepT plasmid, pJG17, overproduce peptidase T by approximately 70-fold. The nucleotide sequence of a 2.5-kb region including pepT has been determined. The sequence codes for a protein of 44,855 Da, consistent with a molecular weight of -46,000 for peptidase T (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration). The N-terminal amino acid sequence of peptidase T purified from a pJG17-containing strain matches that predicted by the nucleotide sequence. A plasmid carrying an anaerobically regulated pepT::lacZ transcriptional fusion contains only 165 bp 5' to the start of translation. This region contains a sequence highly homologous to that identified in Escherichia coli as the site of action of the FNR protein, a positive regulator of anaerobic gene expression. A region of the deduced amino acid sequence of peptidase T is similar to segments of Pseudomonas carboxypeptidase G2, the E. coli peptidase encoded by the iap gene, and E. coli peptidase D.The Salmonella typhimurium pepT gene encodes an aminotripeptidase that is produced at higher levels in cells grown anaerobically than in cells grown aerobically (32). The anaerobic expression of pepT and other Salmonella genes (13, 32) requires the product of oxrA, the Salmonella equivalent of the Escherichia colifnr gene (11,32). The product of fnr is a positive regulator of a family of genes that includes nitrate reductase, fumarate reductase, and nitrite reductase (14,22,27). Most genes regulated byfnr encode components of anaerobic respiratory pathways (29). Peptidase T has no obvious function in growth under anaerobic respiratory conditions, and the significance offnr-dependent regulation of pepT is obscure. As part of an effort to understand the mechanism and physiological significance of the anaerobic regulation of pepT, we have cloned the pepT gene and determined its nucleotide sequence. MATERIALS AND METHODSBacterial strains and plasmids, media, and growth conditions. The bacterial strains and plasmids used in this work are listed in Enzyme assays. 3-Galactosidase activity was determined as described by Miller (19). Peptidase T was assayed, using Met-Gly-Gly as the substrate and high-performance liquid chromatography of reaction mixtures derivatized with trinitrobenzenesulfonyl chloride (33).Purification of peptidase T. Peptidase T was purified from S. typhimurium TN2322 grown without shaking to stationary phase in minimal glucose medium supplemented with Casamino Acids (0.1%) and ampicillin (100 RgIml). The harvested cells (26.4 g) were suspended in buffer (0.01 M Tris-HCl, pH 7.5) and disrupted by sonication. After centrifugation for 1 h at 27,000 x g, the supernatant was diluted to 68 ml with buffer and then 10 ml of protamine sulfate solution (2%) was added with stirring at 4°C over 1 h. Centrifugation at 12,000 x g for 10 min produced a pellet containing the peptidase T activity. This pellet was suspended i...

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“…The enzyme was incubated with 1mM of each metal ion for 30 min at room temperature and relative activities were assayed by the colorimetric ninhydrin method of Yaron and Mlynar using Leu-Pro as substrate. peptidase p 17 ) have evolved from a common ancient protein. 22 ) Resembling the prolidases, aminopeptidase P, a well-known enzyme, is specific for proline residues that are located at the penultimate position from amino termini of peptides and is activated by Mn 2 +.…”

Section: Effects Of Various Reagents and Metal Ions On The Prolidase mentioning

confidence: 99%

Prolidase fromXanthomonas maltophilia: Purification and Characterization of the Enzyme

Suga

Kabashima

Itō

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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Sequencing and High Expression of Aminopeptidase P Gene from Escherichia coli HB101

Cited by 46 publications

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Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Cloning and nucleotide sequence of the anaerobically regulated pepT gene of Salmonella typhimurium

Prolidase fromXanthomonas maltophilia: Purification and Characterization of the Enzyme

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