Cloning, expression, and characterization of aminopeptidase P from the hyperthermophilic archaeon Thermococcus sp. strain NA1.

Abstract

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nepsilon-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100 degrees C. The APP was thermostable, with a half-life of >100 min at 80 degrees C. This study represents the first characterization of a hyperthermophilic archaeon APP.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 1886–1890
0099-2240/06/$08.00?0 doi:10.1128/AEM.72.3.1886–1890.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 3
Cloning, Expression, and Characterization of Aminopeptidase P from
the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1
Hyun Sook Lee,1Yun Jae Kim,1Seung Seob Bae,1Jeong Ho Jeon,1Jae Kyu Lim,1
Byeong Chul Jeong,2Sung Gyun Kang,1and Jung-Hyun Lee1*
Korean Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Korea,1and
Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Korea2
Received 28 July 2005/Accepted 7 December 2005
Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of
a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular
mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the
putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for
catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs
were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP
(Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme
hydrolyzed the amino-terminal Xaa-Pro bond of Lys(N?-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km,
0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2?-,
Mn2?-, or Zn2?-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at
pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study
represents the first characterization of a hyperthermophilic archaeon APP.
Aminopeptidase P (APP, or X-Pro aminopeptidase; EC
3.4.11.9) is a peptidase that specifically removes the N-terminal
amino acids from peptides in which the penultimate residue is
proline (5). Since the time an enzyme with the specificity of
APP was first purified from Escherichia coli (24), APPs have
been characterized from diverse sources, including bacteria
(16), nematodes (15), insects (14), plants (10), and tissues from
several mammalian species (9, 11). While the physiological
role of APP in bacteria is unclear, mammalian APP is involved
in the protein turnover of collagen and the regulation of bio-
logically active peptides, such as substance P and bradykinin (5,
23, 26). It has been shown that APPs from a number of lacto-
coccal strains may contribute to the abolition of bitterness
during the ripening of cheese by participating in peptide deg-
radation following release into the cheese matrix (17).
To date, however, there have been no reports on the prop-
erties of an APP from either an archaeon or a hyperthermo-
phile. With the availability of a generally applicable combina-
tion of conventional genetic engineering and genomic research
techniques, the genome sequences of some hyperthermophilic
microorganisms are of considerable biotechnological interest
because of their heat-stable enzymes, and many extremely
thermostable enzymes are being developed for biotechnolog-
ical purposes (22). Furthermore, recent advances in the
application of molecular biological tools to hyperthermo-
philic archaea, such as gene knockout techniques and effi-
cient transformation systems, could facilitate the study of
hyperthermophilic archaeal gene function and contribute to
an understanding of the physiology of hyperthermophilic
archaea.
To facilitate the search for valuable and extremely thermo-
stable enzymes and to help answer questions concerning the
physiology of hyperthermophilic archaea grown at extremely
high temperatures, we recently isolated a hyperthermophilic
archaeon, Thermococcus sp. strain NA1 (S. S. Bae et al., un-
published data), and its whole genome sequence was deter-
mined (J.-H. Lee et al., unpublished data). Analysis of the
genome information of Thermococcus sp. strain NA1 revealed
an APP gene that is similar to those for other APPs. In the
present study, the gene corresponding to an APP was cloned
and expressed in E. coli. The recombinant enzyme was puri-
fied, and its characteristics were examined.
MATERIALS AND METHODS
Strains and growth conditions. E. coli DH5? was used for plasmid propaga-
tion and nucleotide sequencing. E. coli BL21-CodonPlus(DE3)-RIL cells (Strat-
agene, La Jolla, CA) and plasmid pET-24a(?) (Novagen, Madison, WI) were
used for gene expression. E. coli strains were cultivated in Luria-Bertani medium
at 37°C, and kanamycin was added to the medium at a final concentration of 50
?g/ml.
DNA manipulation and sequencing. DNA manipulations were performed by
standard procedures as described by Sambrook and Russell (19). Restriction
enzymes and other modifying enzymes were purchased from Promega (Madison,
WI). Small-scale preparations of plasmid DNA from E. coli cells were performed
with a plasmid mini kit (QIAGEN, Hilden, Germany). DNA sequencing was
performed with an automated sequencer (ABI 3100), using a BigDye Terminator
kit (PE Applied Biosystems, Foster City, CA).
Cloning and expression of APP-encoding gene. The full-length Thermococcus
sp. strain NA1 APP gene, flanked by NdeI and XhoI sites, was amplified from
genomic DNA by PCR using the following two primers: sense, 5?-CGACCC
GGCATATGCGCCTCAACAAGCTCACTTCTCTG-3?; and antisense, 5?-CTC
CACATCTCGAGCACGATTATCAGCTCCCTCGGTGCC-3?. The italicized
sequences indicate the NdeI site in the sense primer and the XhoI site in the
antisense primer. The amplified DNA fragment was digested with NdeI and
XhoI, the fragment was ligated with NdeI/XhoI-digested plasmid pET-24a(?),
and the resultant recombinant plasmid was used to transform E. coli DH5?. The
recombinant plasmid was introduced into BL21-CodonPlus(DE3)-RIL cells for
expression after sequence confirmation. Overexpression was induced by the
* Corresponding author. Mailing address: Korean Ocean Research &
Development Institute, Ansan P.O. Box 29, Seoul 425-600, Korea.
Phone: 82-31-400-6243. Fax: 82-31-406-2495. E-mail: jlee@kordi.re.kr.
1886

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addition of isopropyl-?-D-thiogalactopyranoside (IPTG) at the mid-exponential
growth phase, followed by incubation for 3 h at 37°C. The cells were harvested
by centrifugation (6,000 ? g, 20 min, 4°C) and resuspended in 50 mM Tris-HCl
buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol. The cells were disrupted
by sonication and centrifuged (20,000 ? g, 1 h, 4°C). The resulting supernatant
was applied to a column of TALON metal-affinity resin (BD Biosciences Clon-
tech, Palo Alto, CA) and washed with 10 mM imidazole (Sigma, St. Louis, MO)
in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, and
APP was eluted with 300 mM imidazole in the buffer. The pooled fractions were
then buffer exchanged with 50 mM Tris-HCl buffer (pH 8.0) containing 10%
glycerol, using a Centricon YM-10 column (Millipore, Bedford, MA).
The protein concentration was estimated from the absorbance at 280 nm,
using an extinction coefficient of 33,710 M?1cm?1. The protein purity was
examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) performed according to a standard procedure.
Enzyme assays. APP-catalyzed hydrolysis of
prolyl-L-prolyl-4-nitroanilide [Lys(Nε-Abz)-Pro-Pro-pNA; Bachem AG, Buben-
dorf, Switzerland] was detected by the release of Lys(Nε-Abz)-OH, which was
monitored at an excitation wavelength of 320 nm and an emission wavelength of
L-lysyl(ε-2-aminobenzoyl)-L-
417 nm at 80°C (21), using an F-2000 fluorescence spectrophotometer (Hitachi,
Tokyo, Japan). The reaction mixture (1 ml) consisted of 50 mM sodium acetate
buffer (pH 5.0), 15 ?M Lys(Nε-Abz)-Pro-Pro-pNA, and 1.2 mM CoCl2. The
reaction was initiated by adding 2 ?g APP. In all routine assays, APP activity was
measured by detecting proline liberated by the hydrolysis of Met-Pro (Bachem
AG) which was dissolved in methanol. The proline concentration was deter-
mined by a modification of the colorimetric ninhydrin method of Yaron and
Mlynar (25). The ninhydrin reagent was prepared by the addition of 3%
(wt/vol) ninhydrin (Sigma) to a mixture of 60% (vol/vol) glacial acetic acid
and 40% (vol/vol) phosphoric acid, followed by a 30-min incubation at 70°C.
The assay mixture for APP (300 ?l), which contained 50 mM sodium acetate
buffer (pH 5.0), 4 mM Met-Pro, and 1.2 mM CoCl2, was incubated at 80°C for
5 min. The reaction was initiated by the addition of the enzyme, the mixture
was incubated at 80°C for a further 5 min, and the reaction was stopped by the
addition of glacial acetic acid (300 ?l) followed by the ninhydrin reagent (300
?l). After being heated at 80°C for 10 min, the solution was cooled on ice, and
the absorption at 515 nm was determined. The amount of released proline
was calculated, using an extinction coefficient of 4,570 M?1cm?1for the
ninhydrin-proline complex. One unit of APP activity was defined as the
FIG. 1. Sequence comparison of Thermococcus sp. strain NA1 aminopeptidase P (TNA1APP), E. coli aminopeptidase P (EcAPP; gi:113751),
P. furiosus prolidase (PfProl; gi:17380168), and E. coli methionine aminopeptidase (EcMetAP; gi:113740). Dashes indicate gaps, and numbers on
the right represent the positions of the last residues in the original sequence. Identical residues among the four enzymes are marked with asterisks,
and residues with conserved substitutions and semiconserved substitutions are marked with colons and dots, respectively. The putative active-site
residues participating in metal ion coordination and proton shuffling are shown in bold and are underlined, respectively.
VOL. 72, 2006CHARACTERIZATION OF AN ARCHAEAL AMINOPEPTIDASE P 1887

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amount of enzyme that liberated 1 ?mol proline min?1under these assay
conditions.
RESULTS
Primary structure of APP gene and expression of recombi-
nant enzyme. Recently, we isolated a hyperthermophilic ar-
chaeon, Thermococcus sp. strain NA1, grown at high temper-
atures of 70 to 90°C. By analyzing the genome sequence, we
found that an open reading frame (ORF) composed of 1,068
bp encodes a protein consisting of 356 amino acids with a
predicted molecular mass of 39,714 Da. Database searches
indicated that the amino acid sequence of TNA1_APP showed
significant similarity to those encoded by the putative APP
genes in the genome sequences of Thermococcus kodakaraensis
KOD1 (75% identity) (6) and Pyrococcus abyssi GE5 (74%
identity) (3) as well as to that of the dipeptidase from Pyro-
coccus horikoshii OT3 (74% identity) (13). Compared with all
the known APPs or prolidases, that is, E. coli APP (26%), the
cytosolic APPs of Drosophila melanogaster (17%), humans
(19%), rats (21%), tomato plants (14%), and Caenorhabditis
elegans (20%), E. coli prolidase (20%), and archaeon Pyrococ-
cus furiosus prolidase (36%), TNA1_APP yielded low amino
acid sequence identities of below 36% (7). Amino acid se-
quence analysis revealed that five divalent metal ligands (two
aspartic acid residues, a histidine residue, and two glutamic
acid residues) and putative proton shuttle sites (three histidine
residues), all originally identified in the crystal structure of
E. coli APP (24), were highly conserved in TNA1_APP (Fig. 1).
Based on this analysis, it seemed likely that the ORF is a
member of a “pita bread-fold” family (1). A Clustal W align-
ment of the amino acid sequences of TNA1_APP, E. coli APP,
P. furiosus prolidase, and E. coli methionine aminopeptidase is
shown in Fig. 1. Because prolidase (proline dipeptidase; EC
3.4.13.9) only cleaves dipeptides with proline at the C terminus
(5), it was necessary to confirm whether TNA1_APP was ca-
pable of hydrolyzing oligopeptides longer than dipeptides to
classify the enzyme as aminopeptidase P.
To address this, the APP gene was amplified by PCR, and
the expressed enzyme was purified from a soluble cell extract
as described in Materials and Methods. An analysis by SDS-
PAGE showed that a 41-kDa protein (Fig. 2), which was the
expected size of the fusion product comprising the 40-kDa
APP protein and a 1-kDa peptide corresponding to -LEH6-
(His tag) at the C terminus, was the major component of the
purified sample.
Biochemical enzyme characterization. The purified recom-
binant protein was tested for activity toward a typical APP
substrate, and it displayed APP activity by hydrolyzing Lys(Nε-
Abz)-Pro-Pro-pNA (data not shown). APP is a peptidase that
specifically removes the N-terminal amino acids from peptides in
which the penultimate residue is proline (5), although in a num-
ber of cases it is unable to hydrolyze a dipeptide with proline in
the C-terminal position (16, 17, 27). To show whether the recom-
binant enzyme was capable of hydrolyzing a dipeptide as well as
Lys(Nε-Abz)-Pro-Pro-pNA, the hydrolyzing activity toward Met-
Pro was also tested, and the enzyme was clearly able to hydrolyze
the Met-Pro dipeptide. A kinetic analysis was conducted using
Met-Pro, and kinetic parameters such as Km(0.96 mM) and kcat
(541 s?1) were calculated from the measured activity.
As shown in Fig. 3A, APP activity toward Met-Pro was
strongly stimulated at high temperatures and showed a tem-
perature optimum of ?100°C, with ?10% of maximal activity
observed at 40 to 50°C. The same pattern was shown with
Lys(Nε-Abz)-Pro-Pro-pNA. The influence of pH on APP ac-
tivity was evaluated by using different buffers in the pH range
of 4 to 8. Interestingly, the optimum pHs of APP activity were
different for Met-Pro (Fig. 3B) and Lys(Nε-Abz)-Pro-Pro-pNA
(Fig. 3C), with values of 5 and 6.5 to 7, respectively. The reason
that the two substrates could be hydrolyzed at different pH values
needs to be understood, and further analysis is under way.
The enzyme’s thermostability was evaluated by incubating
the enzyme in 50 mM sodium acetate buffer, pH 5, at 80 and
90°C. APP was very thermostable, losing enzymatic activity
with half-life values (t1/2) of ?100 min at 80° and 49 min at
90°C (Fig. 4). Interestingly, incubating APP at 80°C appeared
to increase the relative activity up to 20% within 20 min.
The presence of the conserved amino acid residues for metal
binding implied that TNA1_APP would be influenced by the
addition of metal ions. To examine the metal ion requirement
of TNA1_APP, endogenous metal ions were removed by ul-
trafiltration against metal-free Tris-HCl buffer. After incuba-
tion of TNA1_APP with various metal ions (1.2 mM), the
enzyme activity increased in the presence of Co2?, Mn2?, and
Zn2?ions, although a little or no activation was observed with
other divalent cations, such as Ba2?, Ca2?, Cu2?, Fe2?, Mg2?,
and Ni2?. The effects of Co2?, Mn2?, and Zn2?concentrations
on the activity of APP were very different, with maximal activ-
ity at 3 mM CoCl2, 20 mM MnCl2, and 0.4 mM ZnCl2(Fig. 5).
DISCUSSION
This study is believed to be the first to characterize an APP
from either an archaeon or a hyperthermophile. The APP gene
has been predicted by sequence analysis, and its function was
confirmed by biochemical characterization of the recombinant
enzyme expressed in E. coli.
The kinetic parameters calculated by kinetic analysis using
Met-Pro, i.e., Km(0.96 mM) and kcat(541 s?1), showed that the
dipeptide was a competent substrate of TNA1_APP and were
FIG. 2. SDS-PAGE (12%) of the purified enzyme. The molecular
mass standards (lane M) were phosphorylase b (103 kDa), bovine
serum albumin (77 kDa), ovalbumin (50 kDa), carbonic anhydrase
(34.3 kDa), soybean trypsin inhibitor (28.8 kDa), and lysozyme (20.7
kDa). The band corresponding to the enzyme is indicated by the arrow.
1888LEE ET AL.APPL. ENVIRON. MICROBIOL.

Page 4

comparable to those for the recombinant form of P. furiosus
prolidase obtained using the same dipeptide (Km, 3.3 mM; kcat,
525 s?1) (7). However, whereas TNA1_APP was capable of
hydrolyzing tripeptides such as Met-Pro-Gly (data not shown)
and Lys(Nε-Abz)-Pro-Pro-pNA, it is known that P. furiosus
prolidase does not hydrolyze tri- or tetrapeptides (7).
All other APPs from mesophilic sources show optimum ac-
tivities at temperatures up to 55°C (4, 14, 15, 28). Considering
that most proteases and peptidases isolated from Thermococcus
spp. have enhanced activities at moderately high temperatures
of 70 to 85°C, except for a thermostable thiol protease purified
from the extracellular fraction of T. kodakaraensis KOD1 (18),
it is intriguing that TNA1_APP showed an optimum tempera-
ture above 100°C. The fact that TNA1_APP is a hyperthermo-
stable enzyme was also demonstrated by the enzyme having a
t1/2of ?100 min at 80°C. Furthermore, TNA1_APP seemed to
be significantly activated by incubation at high temperatures.
Heat-induced activation over a short period of time has also
been observed with other proteolytic enzymes purified from P.
horikoshii, Sulfolobus solfataricus, and Thermococcus sp. strain
NA1, including an acylamino acid-releasing enzyme (12), in-
tracellular protease (8), prolyl endopeptidase, and carboxy-
FIG. 3. Effects of temperature (A) and pH (B and C) on activity of APP. (A) Activity assays were performed under standard conditions as the
sample temperature was increased from 40 to 100°C. (B and C) Activity assays were performed under standard conditions for the hydrolysis of
Lys(Nε-Abz)-Pro-Pro-pNA (open symbols) and Met-Pro (closed symbols) with the following buffers (each at 50 mM): sodium acetate (circles), pH
4 to 6; KMES [potassium 2-(N-morpholino)ethanesulfonic acid] (squares), pH 5.5 to 7; MOPS (morpholinepropanesulfonic acid) (triangles), pH
6.5 to 7.5; and HEPES (upside-down triangles), pH 7 to 8.
FIG. 4. Thermal inactivation of APP. Semilog plots of the remain-
ing activity versus the incubation time are shown. APP (4 nM) was
incubated at 80°C (E) or 90°C (F) in 50 mM sodium acetate buffer, pH
5, containing 1.2 mM CoCl2. At the times shown, aliquots were re-
moved, and the activity was measured in the same buffer at 80°C, using
Met-Pro as the substrate. The lines were obtained by linear regression
of the data.
FIG. 5. Effects of metal ions on APP activity. Activity assays were
performed under standard conditions while the concentration of diva-
lent metal ions was varied. F, Co2?; ■, Mn2?; Œ, Zn2?.
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peptidase (Lee et al., submitted for publication). Although the
mechanism was not investigated, it can be deduced that a
heat-induced conformational change in the enzyme increased
its activity. Guagliardi et al. (8) are of the view that the stim-
ulation of activity upon preheating is a feature of some en-
zymes from thermophilic sources, and they believe that this
reflects the activation of the low-activity status adopted by
these catalysts at moderate temperatures.
The pH optimum of TNA1_APP was in a mildly acidic range
(pH 5 and 6.5 to 7), which is distinct from other APPs. Most
APPs show optimum activity at pH 7 to 9, although a mem-
brane-bound APP from a bovine lung had a pH optimum of 6.5
for bradykinin hydrolysis (20). Furthermore, the optimum pH
values were different for the hydrolysis of Met-Pro and Lys(Nε-
Abz)-Pro-Pro-pNA, and the reasons for this need to be inves-
tigated.
The APP from Thermococcus sp. strain NA1 appeared to be
a metalloenzyme stimulated by Co2?and Mn2?, like the en-
zyme from Lactococcus lactis (16, 17) and those from other
sources (2, 11). Zn2?also showed a stimulatory effect, as opposed
to the Zn2?inhibition of all mammalian and C. elegans APPs.
We have been able to establish the function of a predicted
ORF in a hyperthermophilic archaeon by combining conven-
tional genetic engineering and genome research techniques.
The hyperthermophilic archaea whose sequences have been
determined in the Thermococcaceae family have genomes con-
sisting of about 2,000 genes, and almost half of the predicted
ORFs are putative or hypothetical (6). Currently, studies on
the physiology and genetics of hyperthermophiles are limited
by a lack of molecular biological tools and by the extreme
growth conditions required. We hope that a genome-wide
approach of the kind used in the present study can contrib-
ute to establishing the function of putative or hypothetical
genes, eventually leading to an understanding of archaeal
physiology.
ACKNOWLEDGMENTS
We express our appreciation to J.-H. Hong and S.-C. Shin from
Macrogen Inc. for valuable information.
This work was supported by the KORDI in-house program
(PE91900) and the Marine and Extreme Genome Research Center
program of Ministry of Maritime Affairs and Fisheries, Republic of
Korea.
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