The active site of carboxypeptidase <i>Taq</i> possesses the active-site motif His–Glu–X–X-His of zinc-dependent endopeptidases and aminopeptidases

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

doi:10.1093/protein/9.6.467

Cited by 23 publications

(14 citation statements)

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“…The MHACGHD sequence in B2 (Fig. 1) was not conserved as the zinc chelation motif of CP (29,37), but was conserved well in PP, CPS, and aminoacylase from B. stearothermophilus. The mechanism of zinc chelation in PP may be a little different from those in the known zincdependent metallocarboxypeptidases.…”

Section: Discussionmentioning

confidence: 99%

“…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: 99%

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confidence: 99%

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Ishikawa¹,

Ishida²,

Matsui³

et al. 2001

Appl Environ Microbiol

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“…The amount of Zn(II) in InsA was determined by inductively coupled plasma atomic emission spectrometry (model SPS‐1200VR; Seiko Instruments), as described previously (see Lee, S.H. et al ., 1996).…”

Section: Methodsmentioning

confidence: 99%

Involvement of two domains with helix–turn–helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats

Ohta¹,

Yoshimura

Ohtsubo³

2004

Molecular Microbiology

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SummaryThe insertion element IS 1 has two open reading frames (ORFs), insA and insB , and produces a transframe protein InsAB, known as IS 1 transposase, by translational frameshifting. The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS 1 transposition. Unless frameshifting occurs, IS 1 produces InsA protein, which also binds to IRs and therefore acts as an inhibitor of transposition, as well as a transcriptional repressor of the promoter in IRL. A helix-turn-helix (HTH) motif present in both transposase and InsA is thought to be involved in IRspecific DNA binding. A comparison of transposases encoded by IS 1 family elements reveals that the Nterminal regions contain four conserved cysteine residues, which appear to constitute a C 2 C 2 zinc finger (ZF) motif. This motif is also thought to be involved in IR-specific DNA binding. In this study, we show that IS 1 transposases with an amino acid substitution in the HTH or ZF motif lose the ability to promote transposition. We also show that transposases, as well as InsA proteins with the same substitution, lose the ability to repress the activity of the IRL promoter, and that purified InsA mutant proteins lose the ability to bind to the IRL-containing fragment. Furthermore, we show that InsA protein co-ordinates Zn(II) with the four cysteine residues as ligands and loses the ability to bind to the IRL-containing fragment in the presence of an agent chelating Zn(II). These findings indicate that IS 1 transposase has two domains with HTH and ZF motifs responsible for IR-specific DNA binding in promoting transposition. It is assumed that the two domains are needed for transposase to bind to each IR in an oriented manner in order to place a catalytic domain in the C-terminal region of the transposase to a region around the IR end, where the strand transfer reaction occurs in a transpososome.

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“…Vol. 61,2004 Multi-author Review 2707 members of the M2 family, ACE2 is a carboxypeptidase rather than a peptidyl dipeptidase, being the first mammalian carboxypeptidase to be identified to contain the HEXXH motif rather than the typical carboxypeptidase A-like motif, HXXE(X) 123-132 H. In this sense, ACE2 more closely resembles the bacterial carboxypeptidases of the M32 family, Thermus aquaticus carboxypeptidase (TaqCP) [46] and Pyrococcus furiosus carboxypeptidase (PfuCP) [47], which also utilize an HEXXH motif for zinc binding [48] (fig. 3a).…”

Section: Catalytic Properties Of Ace2mentioning

confidence: 99%

What’s new in the renin-angiotensin system?

Warner

Smith

Hooper

et al. 2004

CMLS, Cell. Mol. Life Sci.

133

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Angiotensin-converting enzyme-2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein which functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the C-terminus of its substrates. ACE2 efficiently hydrolyses the potent vasoconstrictor angiotensin II to angiotensin (1-7). It is a consequence of this action that ACE2 participates in the renin-angiotensin system. However, ACE2 also hydrolyses dynorphin A (1-13), apelin-13 and des-Arg(9) bradykinin. The role of ACE2 in these peptide systems has yet to be revealed. A physiological role for ACE2 has been implicated in hypertension, cardiac function, heart function and diabetes, and as a receptor of the severe acute respiratory syndrome coronavirus. This paper reviews the biochemistry of ACE2 and discusses key findings such as the elucidation of crystal structures for ACE2 and testicular ACE and the development of ACE2 inhibitors that have now provided a basis for future research on this enzyme.

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The active site of carboxypeptidase Taq possesses the active-site motif His–Glu–X–X-His of zinc-dependent endopeptidases and aminopeptidases

Cited by 23 publications

References 0 publications

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Involvement of two domains with helix–turn–helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats

What’s new in the renin-angiotensin system?

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