Acylamino Acid-releasing Enzyme from the Thermophilic ArchaeonPyrococcus horikoshii*

When the genome of the thermophilic archaeonPyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme’s proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coliwith the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 °C, with its optimum temperature over 90 °C. The specific activity of the enzyme increased 7–14-fold with heat treatment, suggesting the modification of the enzyme’s structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of N α-acylated residues in short peptide sequence analysis at high temperatures.

The acylamino acid-releasing enzyme (AARE) 1 catalyzes the NH 2 -terminal hydrolysis of N ␣ -acylpeptides to release N ␣ -acylated amino acids (1). AARE has been used for removal of N ␣ -acylated residues in protein sequence analysis. Until now, AARE has been isolated only from eukaryotic cells (1)(2)(3)(4) and classified as its own serine protease subfamily (5,6). The physiological role of the enzyme is not clear, although it has been suggested that it affects the processing or sorting of proteins (7,8) in eukaryotic cells. From eukaryotic cells, some AARE genes have already been cloned (9,10). However, the production and expression of AARE from these genes within Escherichia coli have not been carried out.
Pyrococcus horikoshii (OT3) is one of the thermophilic archaea collected from a volcanic vent in the Okinawa trough (11). The optimum growth temperature of this archaeon ranges from 90 to 105°C. Most of the proteins from P. horikoshii are thought to be thermostable and active at high temperature. The size of its genome is about 2 Mb, and the guanine-cytosine content is relatively low. At the National Institute of Technology and Evaluation (Tokyo, Japan), sequencing of this genome is in progress (11). From the genome sequencing in P. horikoshii we found a gene that had some homology with a gene for AARE from pig liver (9,12). Therefore, we cloned the gene from P. horikoshii and attempted to express the enzyme in E. coli and examine the characteristics of the expressed enzyme.
Cloning and Expression of the Gene-The genome of P. horikoshii was sequenced by the method of Kaneko et al. (13). The gene that was homologous to the mammalian gene for AARE was found by BLAST search (14). The gene was amplified by the polymerase chain reactionmethod using two primers with unique restriction sites. Amplification of the gene by polymerase chain reaction was carried out at 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, for 35 cycles using Pfu DNA polymerase. The amplified gene was hydrolyzed by the restriction enzymes and inserted in pET11a cut by the same restriction enzymes. The amplified gene was expressed using the pET11a vector system in the host E. coli BL21(DE3) according to the manufacturer's instructions. The host E. coli BL21(DE3) was transformed by the constructed plasmid. The transformant cell was grown in 2YT medium (1% yeast extract, 1.6% tryptone, and 0.5% NaCl) containing ampicillin (100 g/ml) at 37°C. After incubation with shaking at 37°C until the A 600 reached 0.6 -1.0, the induction was carried out by adding isopropyl ␤-D-thiogalactopyranoside at a final concentration of 1 mM and shaking for 4 h at 37°C. The concentration of the enzyme was determined with Coomassie protein assay reagent (Pierce Chemical Company, Rockford, IL) using bovine serum albumin as the standard protein.
Purification of the Enzyme-After induction, the transformant cells were harvested by centrifugation and disrupted with oxide aluminum in 50 mM Tris-HCl buffer (pH 8.0) containing 0.6 M NaCl. After incubation with DNase I (from bovine pancreas; Sigma) for 30 min at room * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB009494.
§ To whom correspondence should be addressed. Fax: 81-298-54-6151; E-mail: ishikawa@nibh.go.jp 1 The abbreviations used are: AARE, acylamino acid-releasing enzyme; Ac-, N ␣ -acetyl; f-, N ␣ -formyl; pNA, p-nitroanilide; ␣-MSH, ␣-melanocyte-stimulating hormone; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; DMF, N, temperature, the crude extract was heated at 85°C for 30 min. The supernatant obtained by centrifugation was dialyzed against 50 mM Tris-HCl buffer (pH 8.0). The dialyzed sample was loaded on a HiTrap Q column (Pharmacia, Uppsala, Sweden). The column was washed with 50 mM Tris-HCl buffer (pH 8.0) and eluted with a linear gradient (0 -1.0 M NaCl in the same buffer). The fractions that showed protein of a similar molecular mass(70 kDa, SDS-PAGE) calculated from the amino acid sequence were concentrated by a Centricon 10 filter (Amicon Inc., Beverly, MA). The concentrated material was loaded on a HiLoad Superdex 200 column (Pharmacia) and eluted with 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl. The fractions demonstrating only one protein band with a molecular mass of 70 kDa by SDS-PAGE were collected and used for the detailed characterizations of the enzyme.
Molecular Weight Determination-The molecular weight of the enzyme was determined by SDS-PAGE performed on a 4 -15% gradient gel in the Phast System (Pharmacia). Protein bands were visualized by staining with Coomassie Brilliant Blue.
The molecular weight was also determined by high performance liquid chromatography (HPLC) and light-scattering photometry. The HPLC was performed on a Superdex 200 column (Pharmacia), and the elution was carried out using 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl at 1.5 ml/min at room temperature. The eluted protein was detected by its absorbance at 280 nm. The light-scattering photometer was conducted at room temperature with a DLS-700S light-scattering photometry (Otsuka Denshi, Shiga, Japan) calibrated with benzene (15) at 633 nm and analyzed by the method of Kamata and Nakahara (16). Optical clarification was performed with polyvinylidene fluoride filters. The specific refractive index increment (dn/dc) was obtained with a KMX-16 refractometer (Chromatix Inc., Sunnyvale, CA at the same wavelength, calibrated with NaCl solution. Enzyme Assay-The activity of the enzyme was determined using Ac-amino acid-pNA and acylpeptides. The enzyme was incubated at 85°C with the substrates in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and 5% N, N-dimethylformamide (DMF), and the released products were measured. The activity toward the Ac-amino acid-pNAs was calculated using the absorption coefficient ⑀ 406 ϭ 9.91 mM Ϫ1 of pNA released (17). The activity toward the acylpeptides was measured by the detection of the exposed ␣-NH 2 group with the cadmium-ninhydrin colorimetric method (18). The analysis of the products from the peptides was performed by HPLC on an ODS-80Ts column (4.6-mm inner diameter ϫ 25 cm) containing TSK gel (Tosoh, Tokyo, Japan). The flow rate was 0.7 ml/min with 95% water, 5% acetonitrile, and 0.1% trifluoroacetic acid. The activity toward ␣-MSH was examined by a PSQ-1 protein sequencer (Shimazu, Kyoto, Japan) at the custom service center of Takara Shuzo.
1 unit of activity corresponds to the amount of enzyme which catalyzes the hydrolysis of 1 mol of substrate/min.
Measurement of Thermostability-Thermostability of the enzyme was measured by the circular dichroism (CD) and the differential scanning calorimetry (DSC).
CD was measured with a CD spectrometer (model 62A DS) (Aviv Instrument, Lakewood, NJ) utilizing a 5.0-mm path length quartz cell in the far UV region. The scan rate of the temperature was 1 K/min. The measurement was carried out in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl.
The experiments of DSC were performed in a model DSC5100 calorimeter (Calorimetry Sciences Corp., Provo, UT). A scan rate of 1 K/min was used throughout. Before measurement, the sample was dialyzed against 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and degassed with an aspirator for 15 min. Instrument base lines were established with both cells filled with dialysate; the reference cell remained filled with dialysate during the protein scans.

RESULTS AND DISCUSSION
Expression of the Enzyme-In the genome sequenced from P. horikoshii, we found a gene that contained 1,896 base pairs and showed about 20% identity with the AARE gene from pig liver ( Fig. 1) (12). The open reading frame was preceded by AT-rich regions in which a putative ribosome binding site GGTGAT at position Ϫ4 and a putative promoter consensus TTATAT at position Ϫ33 from ATG initiation site were found. This consensus resembles the eukaryotic TATA box and has been confirmed to be the archaeal consensus sequence TT(A/T)(T/A)AX, as determined by analysis of more than 80 archaeal promoters (19). The protein encoded consists of 632 amino acids, making it smaller than AARE (732 amino acids) from a mammal.
However, the proposed active residues (Ser, Asp, and His) of AARE, called "catalytic triad residues" (5, 6, 12), were conserved (Fig. 1). Furthermore, the sequence homology in the Ser, Asp, and His regions (6) of a new family of serine-type peptidases (5) was also observed in this protein. The residues Tyr-492 in the Ser region and Glu-602 in the His region of this protein are not conserved in AARE, but dipeptidyl peptidase (6). These results suggest that this protein, dipeptidyl peptidase, and AARE might be evolutionally related.
The gene was amplified by polymerase chain reaction using two primers. The upper primer (5Ј-TTTTGAATTCTTACATAT-GGGCAAGGGGCTTTCA-3Ј) contained an NdeI I site (underlined), and the lower primer (5Ј-TTTTGGTACCTTT GGATCC TAAGGGTTTAGCTATCCTTT-3Ј) contained a BamHI site (underlined). The amplified gene was inserted in pET11a, and BL21(DE3) was transformed by the constructed plasmid. After induction for 4 h at 37°C, 50 mg of the thermostable 70-kDa protein (as determined by SDS-PAGE) was purified from 2 liters of culture medium. The result of densitometer (data not shown) for SDS-PAGE (Fig. 2) indicated that the purity was about 99%. The purified protein (0.05 mg) in solution was spotted on an Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) and sequenced by a PSQ-1 protein sequencer (Shimazu) at the custom service center of Takara Shuzo. By sequence analysis, the first 20 amino acid residues of the NH 2 terminus except the NH 2 -terminal Met were detected. The NH 2 -terminal sequence was identical to that anticipated from the nucleotide sequence. The extra f-Met residue at the NH 2 terminus of the nascent polypeptide, encoded by the initiation codon, was not detected. This shows that the Gly residue neighboring the starting f-Met residue has a small radius of gyration which is essential for the removal of the f-Met residue to yield the mature enzyme (20), and the soluble protein was processed correctly. The high yield of the recombinant protein indicates the very efficient post-translation of the P. horikoshii gene inside E. coli cells, including the removal of the f-Met residue from the nascent polypeptide. It is suggested that the pET system is a good tool for the production of this protein, and the protein has no toxic effect on the growth of E. coli.
Unlike other AARE, the protein derived from P. horikoshii needed a high concentration of NaCl to be dissolved. Therefore, the purified protein solution used for the characterization contained 0.6 M NaCl. The molecular mass of the purified protein, as determined by SDS-PAGE (Fig. 2), was consistent with that (72,390 Da) calculated from the amino acid sequences. The molecular mass of the protein determined by HPLC was about 150,000 Da (data not shown). The weighted-average molecular weight measured by light-scattering photometry using the dn/dc value determined for chicken gizzard myosin was about 160,000. Therefore, the protein is likely a dimer structure, instead of the four identical subunits found in mammals (9,17). The absorption coefficient (A 280 nm ) of the protein at 1% was determined to be 12.0.
Specificity of the Enzyme-To examine the activity of this protein, we used Ac-Leu-pNA, Ac-Ala-pNA, Ac-Tyr-pNA, Leu-pNA, and Ala-pNA as substrates. Table I shows the hydrolytic activity (releasing of pNA) of the protein for them. At 85°C and pH 5.4, the protein exhibited some hydrolytic activity for Ac-Leu-pNA, Ac-Ala-pNA, Ac-Tyr-pNA and no hydrolytic activity for Leu-pNA and Ala-pNA. As shown in Table I, the protein also had hydrolytic activity for acetylpeptides and formylpeptides. Analysis of the products by HPLC revealed that the protein could only release the acylated amino acids from acylpeptides. Therefore, this protein was concluded to be the AARE from the thermophilic archaeon P. horikoshii. The char-acteristics of this enzyme (hereafter referred to as AAREP) were examined. The optimum pH of AAREP at 85°C was between pH 4.8 and 5.5 (Fig. 3). The optimum temperature of AAREP at pH 5.4 was about 90°C (Fig. 4). Its specificity for small substrates was different from those of AAREs in mammals (1, 17). Unlike the AARE from rat, AAREP released Ac-Leu better than Ac-Ala from Ac-amino acid-pNA; for most of the substrates used, the specific activity of AAREP was higher than that of AARE from rat ( Table I). The activity decreased with increasing the residues of acylpeptides (Table I). Table II shows that AAREP has similar binding affinity for Ac-Leu-pNA, Ac-Ala-pNA, Ac-Ala-Ala, and Ac-Ala-Ala-Ala-Ala. This result is different from that of rat (17). The K m value obtained for Ac-Ala-Ala was a little smaller than that for Ac-Ala-Ala-Ala-Ala (Table II). The active site of AAREP seems to be suitable for relatively short acylpeptides. The hydrolytic activity of AAREP toward ␣-MSH was also examined under the above conditions. The NH 2 -terminal amino acid sequence of ␣-MSH was not detected by the protein sequencer after the incubation with AAREP. This result indicates that AAREP cannot release Ac-Ser from ␣-MSH, unlike the AARE of pig 2 or rat (17). It is peculated that AAREP is able to hydrolyze only short acylpeptides.
Thermostability of the Enzyme-Thermostability of the enzyme was examined with CD and DSC. The CD spectrum in the far-UV region of the enzyme was examined at 25°C and 95°C. The CD spectrum of the enzyme at 95°C was a little different from that at 25°C (Fig. 5). The intensity of the negative ellipticity around 220 nm decreased slightly with increasing temperature. The CD spectrum of AAREP at 95°C was stable for 24 h.  Using DSC from 0°C to 125°C, we measured the heat capacity changes of AAREP. We observed two peaks of heat capacity changes of AAREP over 100°C in the first scan ( Fig.  6A) but no peak in the second scan. Precipitate was observed after the first scan. The temperature of the peaks was independent of the enzyme concentrations examined (0.1-2 mg/ml). This result indicates that the heat inactivation process of the enzyme is irreversible and accompanied by aggregation. The two peaks observed suggest that AAREP consists of two major domains as reported by Miyagi et al. (12).
These results indicate that incubating AAREP at 95°C caused its structure to begin unfolding, but the major conformation of the enzyme remained stable from 0°C to 100°C.
Effect of Heating-After incubating at 95°C, we measured the relative activity of AAREP at 85°C to examine the effect of heating. Incubating at 95°C appeared to increase the relative activity nearly 7-fold (Fig. 7). The enzyme did not lose its    increased activity upon cooling (4 -25°C), suggesting that the activation was irreversible. This heat-activated enzyme (hereafter referred to as HAAREP) was also stable at 95°C for 24 h.
From the light-scattering photometry, the molecular mass of HAAREP was determined to be 260 kDa, and the spatial size of the associated molecule was observed to be expanded in space compared with AAREP; its z-average radius of gyration (R G ) had increased from less than 100 to more than 400. These molecular mass and R G values were virtually constant for nearly 5 days (250 -264 kDa and R G ϭ 416 -445) at room temperature, without significant decomposition of the molecule or development of aggregation. The molecular mass value indicates that the number of monomers constituting the associated molecule averages a little more than 3 in this condition. However, the significant spacial expansion (R G ϭ 416 -445) strongly suggests a conformational change over the whole monomeric structural unit as a result of heat treatment. By heating, the absorbance around 250 -280 nm was increased by 10 Ϯ 1.3%, and the intensity of the negative ellipticity of the CD around 220 nm was decreased slightly (Fig. 5). The changes in the absorbance and CD were parallel to the change in the activity of the enzyme. The NH 2 -terminal sequence of HAAREP remained identical to that of AAREP. The rate of activation by heat treatment was independent of the enzyme concentrations examined (0.04 -1.23 mg/ml). Therefore, it is deduced that the conformational change by heat treatment alters the molecular character of monomer and increases the activity. The NH 2 -terminal section of about 500 residues (12) in the enzyme might be related the conformational change by heat treatment. We are continuing to investigate these points.
In comparing the characteristics of the two enzymes, we found that HAAREP had a higher optimum pH, above 7.0 (Fig.  3) and a higher optimum temperature, 95°C (Fig. 4). The relative specificity of HAAREP for substrates was similar to that of AAREP, although HAAREP showed a 7-14-fold increase in specific activity (Table I). The activation parameters of these enzymes were measured from 50 -85°C (Table III). The temperature dependence on the K m value of Ac-Leu-pNA for AAREP (K m values at 60, 75, 80, 85, and 90°C were 0.689 Ϯ 0.21, 3.60 Ϯ 0.66, 3.85 Ϯ 0.91, 11.0 Ϯ 6.3, and 18.0 Ϯ 5.6 mM, respectively) was similar to that for HAAREP (K m values at 60, 75, 80, 85, and 90°C were 0.876 Ϯ 0.24, 4.12 Ϯ 0.51, 3.99 Ϯ 0.66, 12.9 Ϯ 9.0, and 19.7 Ϯ 2.1 mM, respectively) (Table III). From the temperature dependence on the k cat value of Ac-Leu-pNA, the activation energy of HAAREP was found to be greater than that of AAREP ( Fig. 4 and Table III). Both ⌬S ‡ and ⌬H ‡ values of the activation were increased by heat treatment. The shapes of the DSC curves of HAAREP (Fig. 6B) and AAREP (Fig. 6A) were slightly different from each other, but both enzymes seem to be stable below 100°C. These results suggest that the conformational change in the enzyme by heat treatment has an orienting effect on the catalytic groups of the active site, making the enzyme more active at higher temperatures. It is speculated that HAAREP is a stable intermediate state between the native AAREP state and the heat-inactivated (unfolded) state of the enzyme. Although we have no information about the activity and structure of native AARE in P. horikoshii cells, HAAREP is thought to be the dominant state of the enzyme in P. horikoshii because of the organism's high optimum growth temperature.
Until now, AARE has been found only in eukaryotic cells and thought to be related to the initiation of protein synthesis (1,  7. Activation of the enzyme by heat treatment. AAREP (1.0 mg/ml) was incubated at 95°C in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl. At the time shown, aliquots were taken out, and the activities were measured in the same buffer at 85°C using Ac-Leu-pNA as substrate. Three repetitions were completed to produce the data. The activation parameters (⌬G ‡, E a , ⌬H ‡, ⌬S ‡) and (⌬G, ⌬H, ⌬S) for Ac-Leu-pNA were calculated using typical Arrhenius plots of k cat (Fig.  4. inset) and van't Hoff plots of K m , respectively. [21][22][23]. The existence of this enzyme in the archaeon P. horikoshii suggests that the initiation of protein synthesis in archaea is similar to that in eukaryotic cells. From the fact that a number of eukaryotic intracellular proteins are known to be N ␣ -acylated (24,25), it is speculated that many proteins of archaea are also N ␣ -acylated. Furthermore, the existence of proteasomes in P. horikoshii (26,27) suggests that the action of the enzyme AAREP might be related to the ubiquitin/ATP-dependent system of protein degradation (28 -31). Archaea also contains aminoacylase (26,32), which might play an important role in the recycling of acylamino acids for protein synthesis with the help of AARE.
In eukaryotic cells, a strong degree of genetic similarity between AARE and aminoacylase was suggested by Jones et al. (33). In P. horikoshii, however, the gene for aminoacylase was found at another locus in the genome (27), and AAREP did not share homology or activity with aminoacylase.
AARE from mammals has been used to remove N ␣ -acylamino acid residues from acylpeptides for protein sequencing at relatively low temperature (37°C). AAREP may not be used to remove N ␣ -acylamino acid residues of relatively long acylpeptides. However, AAREP is expected to be used for relatively short acylpeptides in sequence analysis at temperatures higher than 90°C.
Studies about the crystal structure, thermostability, and hydrolytic mechanism of AAREP are in progress.