Characterization of an Aminoacylase from the Hyperthermophilic Archaeon
            <i>Pyrococcus furiosus</i>

Story, Sherry V.; Grunden, Amy M.; Adams, Michael W. W.

doi:10.1128/jb.183.14.4259-4268.2001

Cited by 30 publications

(21 citation statements)

References 67 publications

(74 reference statements)

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“…As shown in Table 1, the order of favorite substrates was Met > Leu > Gln > Glu > Arg > Ala > Asn > Phe = Gly, indicating that Pho ACY has a tendency to exhibit a high activity for amino acids with long side chains. Pfu ACY and Pho CP/ACY also show hydrolase activity against N ‐acetyl‐ l ‐amino acid compounds in the order of preferred l ‐amino acid moieties of Met > Ala = Asn > Glu > Leu for Pfu ACY [12] and Met > Phe > Ala > Trp > Gly for Pho CP/ACY [13], which is rather different from that of Pho ACY.…”

Section: Resultsmentioning

confidence: 99%

“…Some aminoacylases are capable of cleaving dipeptide or tripeptide. Tli ACY can cleave the dipeptide N ‐benzyloxycarbonyl‐Phe‐Gly, and Pho CP/ACY can release the carboxyl‐terminus amino acid residue from di‐, tri‐ and tetrapeptides, whereas Pfu ACY is not able to hydrolyze N ‐formyl‐Met‐Phe, N ‐acetyl‐Met‐Ala and N ‐acetyl‐Met‐Leu‐Phe [12]. Pho ACY can cleave the examined dipeptides Ala‐Ala and Phe‐Ala, but cannot release any amino acid monomer from the tripeptides Ala‐Ala‐Ala and N ‐acetyl‐Ala‐Ala‐Ala (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

et al. 2008

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The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N‐acetyl‐l‐Met, N‐acetyl‐l‐Gln and N‐acetyl‐l‐Leu, but had a lower hydrolytic activity towards N‐acetyl‐l‐Phe. The kinetic parameters Km and kcat were determined to be 24.6 mm and 370 s−1, respectively, for N‐acetyl‐l‐Met at 90 °C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn2+, Mn2+ and Ni2+), and Mn2+ addition caused an alteration in substrate specificity. Site‐directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole‐3‐acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal‐binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

et al. 2008

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“…These results are somewhat different from the metal The activity of enzyme was measured in the presence of metal ions added at 1 mM, unless otherwise noted. One unit of the activity (U) was defined as the amount of enzyme that releases 1 lmol of L-methionine from N-acetyl-L-methionine per min at 90°C effects observed in PfuACY (82% homology to PhoACY) and L-aminoacylase from Thermococcus litoralis (TliACY; 55% homology to PhoACY); namely, Co 2+ enhanced the activity of PfuACY, and Mn 2+ or Ni 2+ resulted in a decrease in the activity of TliACY (Story et al 2001;Toogood et al 2002).…”

Section: Effect Of Metal Ions On Phoacy Activitymentioning

confidence: 99%

“…Metal binding to enzymes plays an important role in their activation and stabilization (Li et al 2005). The activity of a bifunctional aminoacylase/carboxypeptidase from Deinococcus radiodurans R1 was enhanced by metal ions (Lin et al 2007) and a native form of L-aminoacylase from Pyrococcus furiosus (PfuACY) was stabilized in the presence of excess zinc ions (Story et al 2001). The present work describes the effects of metal ions on the activity and thermostability of PhoACY.…”

Section: Introductionmentioning

confidence: 95%

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

et al. 2008

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Recombinant L-aminoacylase (PhoACY) from a hyperthermophilic archeon, Pyrococcus horikoshii, is a zinc-containing metalloenzyme. When the zinc was substituted by Mn(2+) or Ni(2+), its specific activity was significantly increased with acetyl-L-methionine as a substrate. The thermostability of PhoACY was improved when it was incubated with 1 mM Zn(2+), Mn(2+) or Ni(2+). The enzyme with external Zn(2+) addition had no significant loss of the activity when held at 90 degrees C for up to 12 h and moreover had more than a 10-fold longer half-life even at 100 degrees C, compared to the enzyme without Zn(2+) addition. A thermostable structure of the enzyme associated with zinc binding is described based on differential scanning calorimetry.

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“…For example, Pyrococcus furiosus grows optimally at 100°C, utilizing proteins and peptides as substrates, and it produces organic acids, CO 2 , and H 2 . Several enzymes involved in the catabolism of peptides have been purified from P. furiosus, including aminotransferases (4), 2-keto acid oxidoreductases (2,19), glutamate dehydrogenase (1), prolidase (16), acetylcoenzyme A (CoA) synthetases (29), aminoacylase (39), a cobalt-activated carboxypeptidase (11), and pyrollidone carboxypeptidase (42). This list also includes a methionine aminopeptidase (41) and a deblocking aminopeptidase (DAP) (40) as well as another type of aminopeptidase from the related archaeon P. horikoshii (3).…”

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confidence: 99%

Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic ArchaeonPyrococcus furiosus

et al. 2005

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Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ؎ 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co 2؉ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.Aminopeptidases are exopeptidases that catalyze the removal of amino acid residues at the N termini of peptides and proteins. Intracellular proteolytic degradation by aminopeptidases is necessary for modulation of protein concentrations, maintenance of amino acid pools, and removal of damaged proteins (17). In addition, aminopeptidases have more specific functions, including activation (10) and inactivation (21) of biologically active peptides and the removal of N-terminal methionyl residues of newly synthesized proteins. The breakdown of proteinaceous substrates, whether imported into the cell or generated from damaged proteins, are further degraded by di-, tri-, and carboxypeptidases, as well as by aminopeptidases (17). Classification of aminopeptidases has been generally based upon their substrate specificities, such as preference for a neutral, acidic, or basic amino acid in the P1 position of the amino terminus of peptides (44). In recent years, more focus has been placed on classifying peptidases based on structural analyses (35). Aminopeptidases are widely distributed among eukaryotes and prokaryotes, although only a few have been isolated from archaea (37). About two-thirds of all aminopeptidases are represented by the metal-dependent peptidases in which zinc is the most frequently associated metal (34).Hyperthermophilic archaea are potentially rich sources of peptidase-type enzymes, because most of them are capable of using protein-based substrates as ...

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Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Cited by 30 publications

References 67 publications

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic ArchaeonPyrococcus furiosus

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