Purification and Characterization of a Thermostable Carboxypeptidase (Carboxypeptidase<i>Taq)</i>from<i>Thermus aquaticus</i>YT-1

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

doi:10.1271/bbb.56.1839

Cited by 30 publications

(20 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sequence comparison revealed a sequence similarity of approximately 30% and a three-dimensional structure very similar to those of both of these mammalian enzymes (27,29). Another microbial carboxypeptidase with broad substrate specificity was also isolated recently from the thermophilic eubacterium Thermus aquaticus (16). However, no sequence data are so far available for this enzyme.…”

mentioning

confidence: 97%

Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus

et al. 1995

View full text Add to dashboard Cite

Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme.Mammalian metallocarboxypeptidases play key roles in major biological processes, ranging from digestive-protein degradation, as effected by carboxypeptidases A and B (9, 13), to specific proteolytic processing, as in the maturation of biologically active peptides. This latter is the role, for instance, of carboxypeptidase N and enkephalin convertase (10,17).Little is known, however, regarding microbial carboxypeptidases. Recent studies led to the cloning and crystallization of a metallocarboxypeptidase from Thermoactinomyces vulgaris (27,29), which was shown to possess a dual substrate specificity, namely, the capacity to cleave both hydrophobic and basic amino acid residues, combining the activities of carboxypeptidases A and B, respectively. Sequence comparison revealed a sequence similarity of approximately 30% and a three-dimensional structure very similar to those of both of these mammalian enzymes (27,29). Another microbial carboxypeptidase with broad substrate specificity was also isolated recently from the thermophilic eubacterium Thermus aquaticus (16). However, no sequence data are so far available for this enzyme.The lack of any knowledge regarding archaebacterial carboxypeptidases led us to purify and characterize one such enzyme from Sulfolobus solfataricus (3), an extreme thermoacidophilic archaebacterium, isolated from volcanic hot springs, that grows optimally at 87ЊC (7). We found that this protein is a zinc metalloprotease endowed with a unique substrate specificity in that it could release basic, acidic, aromatic, and, to a lesser extent, aliphatic amino acids from artificial substrates. Also, it could withstand temperatures of up to 85ЊC and some organic solvents at up to 40ЊC. Furthermore, we found that salt bridges and the zinc ion play major roles in the kinetic thermal stabilization of this molecule (30). Here, we report the molecular cloning and complete nucleotide sequence of the carboxypeptidase-encoding gene. We show that despite the lack of overall sequence homology with known carboxypeptidases, seven blocks of homology with known metallocarboxypeptidases, spanning the Zn-coordinating histidines, can be identified. We also show that S. solfataricus carboxypeptidase expressed in Escherichia coli is enzymatically...

show abstract

mentioning

confidence: 97%

Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus

et al. 1995

View full text Add to dashboard Cite

show abstract

“…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.…”

mentioning

confidence: 99%

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Ishikawa¹,

Ishida²,

Matsui³

et al. 2001

Appl Environ Microbiol

View full text Add to dashboard Cite

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...

show abstract

“…1) Several thermostable carboxypeptidase 1 genes that span all three kingdoms of life, namely archaea, bacteria, and eucarya, have been identified. Among them, the only three thermostable carboxypeptidase 1 from Thermus aquaticus (carboxypeptidase Taq), 2) T. thermophilus, 3) and the hyperthermophilic archaeon Pyrococcus furiosus 4) have been purified and characterized so far. Thermostable carboxypeptidase 1 is known to be very thermostable, with a temperature optimum of 80-100 C. 2,4) High optimum temperatures for activity have been reported for other carboxypeptidases purified from the bacterium Thermoactinomyces vulgaris 5) and the archaeon Sulfolobus solfataricus, 6) with temperature optima of 60 and 85 C respectively.…”

mentioning

confidence: 99%

“…4) In the case of carboxypeptidase Taq, it cleaves C-terminal neutral, basic, and acidic amino acids most readily, with the exception of Pro and hydrolyses amino acids with long side chains. 2) The biological role of microbial metallocarboxypeptidases has not been established, while mammalian metallocarboxypeptidases play key roles in major biological processes, ranging from digestive-protein degradation, as effected by carboxypeptidase A and B, 7,8) to specific proteolytic processing, as in the maturation of biologically active peptides, as effected by carboxypeptidase N and enkephalin convertase. 9,10) It has been suggested that the broad specificities of P. furiosus CP against synthetic and natural peptide substrates might play a role in digestion and protein turnover as opposed to specific post-translational modification as seen in higher organisms.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Lee¹,

Kim²,

Bae³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

Purification and Characterization of a Thermostable Carboxypeptidase (CarboxypeptidaseTaq)fromThermus aquaticusYT-1

Cited by 30 publications

References 8 publications

Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus

Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

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

Contact Info

Product

Resources

About