CarboxypeptidaseTaq, a Thermostable Zinc Enzyme, fromThermus aquaticusYT-1: Molecular Cloning, Sequencing, and Expression of the Encoding Gene inEscherichia coli

Lee, Sang-Hyeon; Taguchi, Hayao; Yoshimura, Etsuro; Minagawa, Etsuo; Kaminogawa, Shuichi; Ohta, Takao; Matsuzawa, Hiroshi

doi:10.1271/bbb.58.1490

Cited by 42 publications

(21 citation statements)

References 8 publications

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“…Weak activation of PP by cobalt or manganese ions similar to that of some zinc-dependent metallocarboxypeptidases has also been observed (26,28). The results indicate that PP is one of the zinc-dependent metalloenzymes.…”

Section: Discussionmentioning

confidence: 56%

“…A thermostable CP is useful for high-temperature analysis of the C-terminal amino acid sequences of proteins. Recently, several thermostable CPs from the thermophilic bacteria Thermoactinomyces vulgaris (35,36) and Thermus aquaticus (27,28,29) and the thermophilic archaea Sulfolobus solfataricus (12,13,38) and Pyrococcus furiosus (8) have been characterized. Using genome sequencing for P. horikoshii (20, 21), we found two kinds of genes encoding CP-homologous proteins.…”

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confidence: 99%

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Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Ishikawa¹,

Ishida²,

Matsui³

et al. 2001

Appl Environ Microbiol

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Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.Pyrococcus horikoshii OT3 is one of the thermophilic archaea collected from a volcanic vent in the Okinawa Trough (16,20,21). This archaeon's optimum growth temperature ranges from 90 to 105°C. Most of the proteins from P. horikoshii are expected to be thermostable and active at high temperatures.Carboxypeptidase (CP) (peptidyl-L-amino-acid hydrolase; EC 3.4.12.1), which hydrolyzes the peptide bond at the C termini of peptides and proteins, is widely distributed in many organisms. Mammalian carboxypeptidase A (CPA) and CPB have been studied in detail (1,11,24). A thermostable CP is useful for high-temperature analysis of the C-terminal amino acid sequences of proteins. Recently, several thermostable CPs from the thermophilic bacteria Thermoactinomyces vulgaris (35,36) and Thermus aquaticus (27,28,29) and the thermophilic archaea Sulfolobus solfataricus (12,13,38) and Pyrococcus furiosus (8) have been characterized. Using genome sequencing for P. horikoshii (20, 21), we found two kinds of genes encoding CP-homologous proteins. One protein has 40% identity to the CP of T. aquaticus (28). The other has approximately 45% identity to the CP of S. solfataricus (13) and aminoacylase (N-acyl-L-amino acid amidohydrolase; EC 3.5.1.14) (3) of Bacillus stearothermophilus (33). In the latter protein, some of the amino acids which corresponded to the essential acti...

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Section: Discussionmentioning

confidence: 56%

mentioning

confidence: 99%

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Ishikawa¹,

Ishida²,

Matsui³

et al. 2001

Appl Environ Microbiol

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“…NA1, growing at high temperatures (70-90 C), and, by analyzing the genome sequence, we found that the open reading frame, composed of 1,497 bp, encodes a protein homologous to thermostable carboxypeptidase 1, which were purified and characterized from P. furiosus DSM 3638 (84% identity), 15,16) T. aquaticus YT-1 (35% identity), 17) and T. thermophilus HB27 (35% identity) 3) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic ArchaeonThermococcussp. NA1

Lee¹,

Kim²,

Bae³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…The other ORF exhibited highest homology of 73% with that of P. horikoshii. Carboxypeptidase Taq from Thermus aquaticus (Lee et al, 1994), Thermus thermophilus (Nagata et al, 2004), Thermococcus sp NA1 (Lee et al, 2006) and P. furiosus (Chang et al, 2001) have been characterized. Carboxypeptidase from Thermococcus sp NA1 shows broad substrate specificity which makes it a potential candidate in C-terminal sequencing.…”

Section: Carboxypeptidasesmentioning

confidence: 99%

Proteolytic inventory of Thermococcus kodakaraensis

Rasool¹,

Rashid²,

Bashir³

et al. 2013

Afr. J. Microbiol. Res.

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Proteolysis is a very crucial process for development and survival of the cell. Genome sequence data can be employed to estimate proteolysis inventories of different organisms. In this review, we exploit genome sequence data of hyperthermophilic archaeon Thermococcus kodakaraensis to have an overview of the proteolysis in this microorganism. The overview is based on those peptidases that have been characterized, and on putative peptidases that have been identified but have yet to be characterized. In contrast to bacteria, the number of proteolytic enzymes in archaea is quite low. By analyzing the genome sequence data, we tried to establish how T. kodakaraensis maintains its life cycle and other processes by using such a small number of protein scavengers while harboring at high temperature where chances of protein denaturation are high.

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CarboxypeptidaseTaq, a Thermostable Zinc Enzyme, fromThermus aquaticusYT-1: Molecular Cloning, Sequencing, and Expression of the Encoding Gene inEscherichia coli

Cited by 42 publications

References 8 publications

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic ArchaeonThermococcussp. NA1

Proteolytic inventory of Thermococcus kodakaraensis

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