PNP-2 are homotrimers, with a subunit molecular mass of Keywords CXC motif; 5¢-deoxy-5¢-methylthioadenosine phosphorylase; disulfide bonds; hyperthermostability; purine nucleoside phosph
Trang 1mammalian-like purine nucleoside phosphorylase
from the Archaeon Pyrococcus furiosus
Giovanna Cacciapuoti1, Sabrina Gorassini1, Maria Fiorella Mazzeo2, Rosa Anna Siciliano2,
Virginia Carbone2, Vincenzo Zappia1and Marina Porcelli1
1 Dipartimento di Biochimica e Biofisica ‘F Cedrangolo’, Seconda Universita` di Napoli, Italy
2 Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy
Purine nucleoside phosphorylase (PNP) catalyzes the
reversible phosphorolytic cleavage of the glycosidic
bond of purine nucleosides to produce
ribose-1-phos-phate and a free purine base [1–3] PNPs have been
characterized in a variety of species and may be
grouped into two main groups, PNP-1 and PNP-2 PNP-1 are found in prokaryotes, are homohexamers with a subunit of 26 kDa and recognize both 6-oxo and 6-amino purine nucleosides as substrates PNP-2 are homotrimers, with a subunit molecular mass of
Keywords
CXC motif; 5¢-deoxy-5¢-methylthioadenosine
phosphorylase; disulfide bonds;
hyperthermostability; purine nucleoside
phosphorylase
Correspondence
G Cacciapuoti, Dipartimento di Biochimica e
Biofisica ‘F Cedrangolo’, Seconda Universita`
di Napoli, Via Costantinopoli 16, 80138,
Napoli, Italy
Fax ⁄ Tel: +39 081 5667519
E-mail: giovanna.cacciapuoti@unina2.it
(Received 2 February 2007, revised 6 March
2007, accepted 12 March 2007)
doi:10.1111/j.1742-4658.2007.05784.x
We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP) The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5¢-deoxy-5¢-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family mem-bers Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine PfPNP shares 50% identity with MTAP from P furiosus (PfMTAP) The alignment of the protein sequences of PfPNP and PfM-TAP indicates that only four residue changes are able to switch the specif-icity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization PfPNP is highly thermophilic with an optimum temperature of 120C and is characterized
by extreme thermodynamic stability (Tm, 110C that increases to 120 C
in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100C), and remarkable SDS-resistance Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasub-unit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation The characterization of the thermal properties of the C254S⁄ C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability
Abbreviations
hMTAP, human methylthioadenosine phosphorylase; MTA, methylthioadenosine; MTAP,
5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PfPNP, purine nucleoside phosphorylase from P furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from S solfataricus.
Trang 230 kDa and accept only guanosine and inosine as
substrates [3–5] It is interesting to note that many
organisms that express PNP-1 also express PNP-2 [5]
PNP is a ubiquitous enzyme of purine metabolism
that functions in the salvage pathway of cells In
addi-tion to the intrinsic biochemical significance, PNP plays
an important biomedical role In fact, human PNP is a
target for T-cell-related cancers and autoimmune
dis-eases [6] Moreover, differences in substrate specificity
between Escherichia coli PNP and the human enzyme
have been employed for the development of
tumor-directed gene therapy [5,7–10] In this strategy, tumor
cells transfected with E coli PNP gene are able to
convert relatively nontoxic prodrugs into
membrane-permeant cytotoxic compounds To reduce the toxicity
of prodrugs currently used with E coli PNP, a good
experimental approach could be the identification of
PNPs with new substrate specificities In this light,
studies on the molecular and structural characterization
of PNPs from hyperthermophilic Archaea could be
useful to improve the tumor-directed gene therapy
based on the activation of nucleoside analogs prodrugs
Hyperthermophilic Archaea are of extreme
biotechno-logical interest not only for the exceptional stability of
their biomolecules but also for the peculiar substrate
specificity of their enzymes that provide unique models
for studying and understanding enzyme evolution in
terms of structure, specificity and catalytic properties
[11–15] In recent years, the increasing number of
solved crystallographic structures has highlighted the
presence of disulfide bonds in several
hyperthermo-philic proteins [16–20], suggesting that disulfide bond
formation represents a significant molecular strategy
adopted by cytosolic hyperthermophilic proteins to
reach higher levels of thermostability
In Archaea, three enzymes belonging to the PNP
fam-ily have recently been isolated and characterized from
the hyperthermophilic microorganisms Sulfolobus
solfa-taricus(Ss) and Pyrococcus furiosus (Pf) These enzymes
are classified as 5¢-deoxy-5¢-methylthioadenosine
phos-phorylases, as they are able to catalyze the
phosphoroly-tic cleavage of 5¢-deoxy-5¢-methylthioadenosine (MTA),
a natural sulfur-containing nucleoside formed from
S-adenosylmethionine mainly through polyamine
bio-synthesis [21,22] The three enzymes,
5¢-deoxy-5¢-methyl-thioadenosine phosphorylase from S solfataricus
(SsMTAP), 5¢-deoxy-5¢-methylthioadenosine
phosphor-ylase II from S solfataricus (SsMTAPII) and
5¢-deoxy-5¢-methylthioadenosine phosphorylase from P furiosus
(PfMTAP) show features of exceptional thermophilicity
and thermostability with temperature optima and
melting temperatures >100C [23–25] and are
stabil-ized by disulfide bonds [16,20,26] SsMTAP, which
shows a significant sequence identity with E coli PNP,
is a hexamer consisting of six identical subunits of 26.5 kDa and utilizes inosine, guanosine, adenosine, and MTA as substrates [23] The crystal structure of SsMTAP reveals that it contains three intermonomer disulfide bridges in each hexamer [16] SsMTAP II is a homohexamer (subunit 30 kDa), characterized by extre-mely high affinity towards MTA SsMTAPII shares 51% identity with human 5¢-deoxy-5¢-methylthioadeno-sine phosphorylase (hMTAP) and is able to recognize adenosine [24] in contrast to hMTAP, which is highly specific for MTA The crystal structure of SsMTAPII indicates a dimer of trimers with two pairs of intrasub-unit disulfide bridges [20] Finally, PfMTAP is a hexa-meric protein that, like SsMTAPII, shares 50% identity with hMTAP PfMTAP is characterized by a broad sub-strate specificity with 20-fold higher catalytic efficacy for adenosine and MTA than for inosine and guanosine [25] PfMTAP is stabilized by two intrasubunit disulfide bridges [26]
The analysis of the complete genomic sequence of
P furiosus shows, beside PfMTAP, a second enzyme that, on the basis of the high identity with PfMTAP is annotated as MTAPII We renamed this enzyme as PNP as it is completely unable to cleave MTA while, in analogy with human PNP, it is characterized by a strict substrate specificity towards inosine and guanosine This paper describes the cloning, recombinant expression and structural and functional characteri-zation of purine nucleoside phosphorylase from the hyperthermophilic archaeon P furiosus (PfPNP) aimed
to elucidate the structure⁄ function ⁄ stability relation-ship in this enzyme and to explore its biotechnological applications By integrating classical biochemical meth-odologies with mass spectrometry, we assigned three intrasubunit disulfide bridges important for the enzyme stability Finally, the characterization of the thermal properties of the C254S⁄ C256S mutant allowed us to propose that the CXC motif in the C-terminal region
of PfPNP may also account for the extreme thermo-stability of the enzyme PfPNP, on the basis of its substrate specificity is the first example of a mamma-lian-like PNP reported in Archaea
Results and Discussion
Analysis of PfPNP gene, primary sequence comparison and expression
The analysis of the complete sequenced genome of
P furiosus revealed an open reading frame (PF0853) encoding a 265-amino acid protein homologous to hMTAP This enzyme is annotated as hypothetical
Trang 3MTAPII and has been renamed by us PfPNP The
putative molecular mass of the protein predicted from
the gene was 29 208 Da The coding region starts with
an ATG triplet at the position 826577 of the P
furio-sus genome The first stop codon TAG is encountered
at the position 827374 Upstream from the coding
region 24 bp before the starting codon there is a
stretch of purine-rich nucleosides (CCTCC) that may
function as the ribosome-binding site [27] Putative
promoter elements, which are in good agreement with
the archaeal consensus [27] designed box A and box B
are found close to the transcription start site A
hexa-nucleotide with the sequence TATTATA similar to the
box A is located 19 bp upstream from the start codon
and resembles the TATA box which is involved in
binding the archaeal RNA polymerase [27] A putative
box B (ATGC) overlaps the ATG codon Finally, a
pyrimidine-rich region (TTTTTAT) strictly resembling
the archaeal terminator signal [27], is localized 8 bp
downstream from the translation stop codon
To overproduce PfPNP, the gene was amplified by
PCR and cloned into pET-22b(+) under the T7 RNA
polymerase promoter The gene sequence was found to
be identical with the published sequence [28] except for
a single mutation at the third codon, where A was
sub-stituted with G resulting in Arg instead of Gly Since
in repeated gene amplification experiments carried out
utilizing different preparation of the same primers we
always obtained the same result, it is possible to
hypo-thesize that a mistake is present in GenBank at level
of the third codon of PfPNP gene Comparison of
the deduced primary sequence of PfPNP with
enzy-mes present in GenBank Data Base reveals a much
higher similarity of PfPNP with members of MTAP
family, such as MTAP from Pyrococcus abyssi (87%
identity), MTAP from Pyrococcus horikoshii (84%
identity), MTAP from Thermococcus kodakarensis
(76% identity), than with members of PNP family such
as PNP from Methanopyrus kandlery AV19 (51%
iden-tity) and PNP from Aquifex aeolicus (47% ideniden-tity)
This evidence could also be noted by comparing the
amino acid sequence of PfPNP with related enzymes
characterized from various sources, that indicated a
high sequence identity with PfMTAP (50%),
SsMTAP-II (48%) and hMTAP (40%) while a lower identity
was observed with E coli PNPII (30%) and hPNP
(27%) No significant similarity was found with E coli
PNP, SsMTAP, and PNP from Thermus thermophilus
The recombinant PfPNP was produced in a soluble
form in E coli BL21 cells harboring the plasmid
pET-PfPNP at 37C in the presence of
isopropyl-b-d-thio-galactoside Under the experimental conditions selected
for the expression, about 10 g of wet cell paste was
obtained from 1 L of culture The PfPNP activity of recombinant E coli BL21 cells harboring pET-PfPNP, was 17.9 unitsÆmg)1 at 80C, confirming that PfPNP gene had been cloned and expressed
Enzyme purification and properties Recombinant PfPNP was purified to homogeneity by a fast and efficient two-step procedure that utilizes a heat treatment and affinity chromatography on MTI-Sepharose (Table 1) SDS⁄ PAGE of PfPNP reveals a single band with a molecular mass of 29 ± 1 kDa, which is in fair agreement with the expected mass cal-culated from the amino acid sequence The identity of the protein was checked by N-terminal sequencing which also revealed that the initial methionine was post-translationally removed This result was con-firmed by MALDI-MS analysis of the HPLC purified protein The experimental mass value (m⁄ z 28 966.23) was in good agreement with the theoretical average molecular mass of the full length gene product without the N-terminal methionine (28 977.39 Da), being the observed mass difference partly due to the presence of disulfide bridges
The molecular mass of PfPNP was estimated to be
180 ± 9 kDa by size exclusion chromatography, which indicated a hexameric structure in solution Therefore,
on the basis of its quaternary structure PfPNP is a mem-ber of the hexameric group of PNPs (PNP-1) together with the structurally characterized PNPs from Archaea, including SsMTAP [16,23], SsMTAPII [20,24], and PfMTAP [25,26] and from Bacteria, such as PNP from E coli (EcPNP) [29], PNP from T thermophilus (TtPNP) [30], and E coli uridine phosphorylase [31]
Substrate specificity and comparative kinetic characterization
To elucidate the physiological role of PfPNP and its functional relationships with PfMTAP, we carried
Table 1 Purification of recombinant purine nucleoside phosphory-lase from P furiosus A typical purification from 10 g of wet cells is shown.
Total protein (mg)
Total activity (units)
Specific activity a (unitsÆmg)1)
Yield (%) Purification (n-fold)
a Specific activity is expressed as nmol of hypoxanthine formed per min per mg of protein at 80 C.
Trang 4out a detailed kinetic characterization of PfPNP and a
comparative kinetic analysis of the two enzymes
Initial velocity studies carried out with increasing
concentrations of purine nucleosides in the presence of
saturating concentration of phosphate gave typical
Michaelis–Menten kinetics While PfMTAP showed a
broad substrate specificity being able to
phosphorolyti-cally cleave both 6-amino and 6-oxo purine nucleosides
[25], PfPNP, in analogy with mammalian enzyme, is
specific for guanosine and inosine with Km values of
122 and 322 lm, respectively Moreover, the relative
efficiency of the nucleoside substrates was determined
by comparing the respective kcat⁄ Kmratios As shown
in Table 2, the substrate activity of PfPNP with
ino-sine and guanoino-sine gave comparable kcat⁄ Km values
(2.61· 107 and 2.2· 107, respectively) that are four
orders of magnitude higher than those of PfMTAP for
the same substrates, indicating that PfPNP is the
enzyme physiologically involved in the 6-oxo-purine
nucleoside catabolism in P furiosus When phosphate
concentration was varied at fixed saturating
concentra-tion of inosine, non-Michaelis–Menten kinetics were
observed with two different Km values for phosphate
of 6.2 and 259 lm This result is in agreement with the data reported in the literature on the complexity of phosphate binding for PfMTAP [25] and for PNPs from various sources [32,33]
The results of substrate specificity studies are supported by the analysis of the sequence alignment
of PfPNP, PfMTAP, hMTAP and hPNP reported
in Fig 1 The amino acid residues of PfPNP and
Table 2 Kinetic parameters of PfPNP and PfMTAP Activities were determined at 80 C as described in Experimental procedures.
K mapp (l M ) k cat (s)1) k cat ⁄ K m app (s)1Æ M )1) PfPNP
PfMTAP a
a The data for PfMTAP have already been published [25].
Fig 1 Multiple sequence alignment of PfPNP, PfMTAP, hMTAP, and hPNP The phosphate (w) ribose, (m) and base (d) binding sites of hMTAP (above the sequence) and of hPNP (below the sequence) are indicated Identical residues between PfPNP and PfMTAP at the hypo-thetical active sites are highlighted in a grey box PfPNP cysteine residues are shown in white lettering on a black background.
Trang 5PfMTAP corresponding to those present at the active
sites of hPNP [34] and hMTAP [35], respectively, were
compared with highlight the changes that may account
for the difference in substrate specificity among the
two P furiosus enzymes As expected on the basis of
the very high sequence identity (50%), the hypothetical
active sites of PfPNP and PfMTAP are very similar
and only few key residue changes are observable
Three important substitutions are localized at the level
of the base binding site where Glu169, Asn211, and
Ala213 of PfPNP replace Ser163, Asp204 and Asp206
of PfMTAP, respectively It is important to note that
these substitutions are exactly those that are
respon-sible for the different substrate specificity of hPNP and
hMTAP (Glu201, Asn243 and Val245 of hPNP instead
of Ser178, Asp220 and Asp222 of hMTAP,
respect-ively) The last important substitution is observable at
the ribose pocket where His223 of PfPNP substitutes
Ala215 of PfMTAP Also in this case, the same
substi-tution takes place in mammalian enzyme where the
change of His257 of hPNP with Val233 of hMTAP
makes hydrophilic the hydrophobic pocket, preventing
the binding of the 5-methylthioribose moiety As for
the remaining differences between the hypothetical
active sites of the two P furiosus enzymes, they are all
conservative substitutions except for the change of
Ile56 of PfPNP with Phe57 of PfMTAP It is
interest-ing to note in this respect that the correspondinterest-ing
resi-due Tyr88 of hPNP is not determinant since the
interactions between PNP and sugar ring are primarily
hydrophobic [34] In conclusion, only four
substitu-tions are able to switch the specificity of the enzyme
from 6-oxo to 6-amino purine nucleoside
phosphory-lase still maintaining the same overall active site
organ-ization On the basis of the reported results, PfPNP
shows peculiar structural and functional properties
The enzyme, in fact, although characterized by the
hexameric quaternary structure distinctive of bacterial
PNP, exhibits a substrate specificity that makes it the
first archaeal mammalian-like PNP
Thermal properties and limited proteolysis
The temperature dependence of the activity of PfPNP in
the range from 30C to 140 C is shown in Fig 2 The
enzyme is highly thermoactive; its activity increased
sharply up to the optimal temperature of 120C and a
50% activity was still observed at 133C This behavior
led to a discontinuity in the Arrhenius plot at about
84C, with two different activation energies
To study the thermodynamic stability of PfPNP we
measured the residual activity after 10 min incubation
at increasing temperature The corresponding diagram
reported in Fig 3A is characterized by a sharp trans-ition that allowed us to calculate an apparent melting temperature of 110 C This value increases to 120 C
in the presence of 100 mm phosphate indicating that this substrate is able to stabilize the enzyme toward temperature A similar substrate protection against thermal denaturation was also observed for the homol-ogous enzymes SsMTAP [23], PfMTAP [25],
SsMTAP-II [24], and hMTAP [36]
The resistance of PfPNP to irreversible heat inacti-vation processes was monitored by subjecting the enzyme to prolonged incubations in a temperature range from 100 to 115 C and by measuring the resid-ual activity under standard conditions As observed in Fig 3B, the enzyme decay obeys first-order kinetics The results obtained indicate that PfPNP is character-ized by a notably high kinetic stability retaining full activity after 4 h incubation at 100 C (inset in Fig 3B) and showing half-lives of 69, 12, and 5 min at
105, 110, and 115C, respectively Kinetic stability has been reported as a property of some naturally occur-ring proteins that are trapped in their native conforma-tions by an high energy barrier that slows down the unfolding processes It has also been reported in the literature that kinetically stable proteins are extremely resistant to SDS-induced denaturation [37] Therefore,
we incubated PfPNP in the presence of 2% SDS at increasing temperature and then we measured the cata-lytic activity under standard conditions As shown
in Fig 4A, PfPNP remains fully active after 30 min
0 0 0 0
0 1
0 1 0 1 0 0
0 0
) C
° ( e u t a r p m e T
0 x T /
2 3 4 5
0 3 0 3 0 2
Fig 2 The effect of temperature on PfPNP activity The activity observed at 120 C is expressed as 100% The assay was per-formed as indicated under Experimental procedures Arrhenius plot
is reported in the inset; T is measured in Kelvin.
Trang 6incubation at 50C and still retains 60% residual
activity after 5 min incubation at 90C Phosphate is
able to increase the already high stability of PfPNP
toward the detergent In fact, after 15 min incubation
at 100C with 2% SDS and 100 mm phosphate, the
enzyme still shows about 20% residual activity
(Fig 4B) while in the same experimental conditions
but in the absence of phosphate, it appears completely
inactive It is interesting to note that no protective
effect against SDS inactivation has been observed in
the presence of inosine indicating that only phosphate
is able to form a binary complex with the enzyme
These results suggest that PfPNP, in analogy with
PfMTAP [26], could act via an ordered Bi-Bi
mechan-ism with the phosphate binding preceding the
nucleo-side binding in the phosphorolytic direction
The high kinetic stability of PfPNP is indicative of a
compact and rigid structure that allows the protein to
retain its native state in extreme experimental
condi-tions It has been proposed that kinetic stability, by
lim-iting the access of the protein to partially and globally unfolded conformations could be responsible not only for the extreme resistance to SDS-induced denaturation but also for the stability against proteolytic degradation [37] To verify this hypothesis and to obtain information about the flexible regions of PfPNP exposed to the sol-vent and susceptible to proteolytic attack we subjected the enzyme to limited proteolysis PfPNP resulted com-pletely resistant to several proteases, such as trypsin, chymotrypsin, proteinase K and subtilisin Only ther-molysin was able to cleave the enzyme Therefore, pro-teolytic degradation of PfPNP was investigated by measuring the residual activity after incubation with thermolysin at 60C followed by SDS ⁄ PAGE of the digested material A protein band with an apparent molecular mass of about 2.6 kDa less than that of PfPNP appears as the proteolysis proceeds while no concomitant decrease of catalytic activity was observed The analysis of the proteolytic fragment by Edman deg-radation showed that the amino terminus was preserved
Fig 3 Thermostability of PfPNP (A)
Resid-ual PfPNP activity after 5 min of incubation
at temperatures shown in the absence (d)
or in the presence of 100 m M phosphate
(j) Apparent Tms are reported in the inset.
(B) Kinetics of thermal inactivation of PfPNP
as a function of incubation time The
enzyme was incubated at 100 C (see
inset), 105 C (j), 110 C (m), and 115 C
(d) for the time indicated Aliquots were
then withdrawn and assayed for the activity
as described under Experimental
proce-dures.
Fig 4 Effect of phosphate on the thermostability of PfPNP in the presence of 2% SDS (A) The enzyme was incubated at 50 C (s), 70 C (m), 80 C (j), and 90 C (d) with 2% SDS (B) The enzyme was incubated at 80 C (j), 90 C (d), and 100 C (D) with 2% SDS in the presence of 100 m M phosphate At the time indicated, aliquots were withdrawn and assayed for PfPNP activity as described under Experi-mental procedures Activity values are expressed as percentage of the time-zero control (100%).
Trang 7thus indicating that the proteolytic cleavage site is
locali-zed in the C-terminal region Moreover, the observation
that no decrease of enzymatic activity occurred during
proteolysis suggests that the C-terminal peptide of
PfPNP is not necessary for the integrity of the active
site No substrate protection against proteolysis was
observed, confirming the conclusions drawn from the
analysis of the sequence alignment reported in Fig 1
that highlights the absence of hypothetical
substrate-binding sites in the C-terminal region of PfPNP
Effect of reducing agent and disulfide bond
assignment
In recent years, it has becoming evident that, in spite
of their susceptibility to oxidative degradation, cysteine
residues are abundant in genomes of various
hyper-thermophilic Archaea and Bacteria [38] Moreover,
disulfide bonds are now known to occur in many
hyperthermophilic and intracellular archaeal proteins
[16–20], where they are thought to represent an
important structural mechanism to obtain higher
sta-bility The unusual stability features of PfPNP and the
elevated content of cysteine residues deduced from the
gene (six per subunit) prompted us to investigate on
the presence of stabilizing disulfide bonds Therefore,
the thermal stability of PfPNP was investigated by
heating the enzyme in the presence of reducing agents
As reported in Fig 5, after 1 h incubation at temper-atures until 70C, the enzyme remains completely stable even at high concentrations of dithiothreitol (0.8 m) whereas it becomes susceptible to the effect of the reducing agent as the temperature raises In fact, in the presence of 0.4 m dithiothreitol, PfPNP retains only 20% activity after 1 h incubation at 100 C These results offer convincing evidence that PfPNP, in analogy with the homologous PfMTAP, contains disul-fide bonds important for the stability against thermal unfolding and denaturation This hypothesis is suppor-ted by the observation that (a) five out of six cysteine residues of PfPNP are well conserved with respect to PfMTAP (Fig 1), and (b) in PfMTAP four of these cysteine residues are involved in disulfide bonds [26]
To elucidate the S–S bridge arrangement, PfPNP was initially subjected to CNBr reaction and analyzed
by MALDI-TOF-MS both in linear and in reflectron positive-ion mode The signal at m⁄ z 3761.25 generated from the C-terminal peptide 231–265 (monoisotopic molecular mass 3762.14 Da), occurred two mass units lower than expected on the basis of its amino acid sequence, thus indicating the presence of an intrapep-tide disulfide bond joining Cys254 and Cys256 More-over, the signal at m⁄ z 13893.61 was assigned to a three peptides cluster, consisting of peptides 92–187 (average molecular mass 10838.37 Da), 188–201 (aver-age molecular mass 1555.87 Da) and 202–216 (aver(aver-age molecular mass 1499.81 Da) held together by two disulfde bonds (Table 3)
In order to confirm the presence of the Cys254– Cys256 bridge, the peptide mixture originated from CNBr reaction was subjected to enzymatic digestion with Endoproteinase Glu-C In the MALDI-TOF mass spectrum the signal at m⁄ z 3160.80 corresponded to the peptide 236–265 containing the S–S bridge (mono-isotopic molecular mass 3159.81 Da) Nevertheless, isotope distribution of the signal could suggest the presence of a low percentage (10%) of the peptide having the cysteine residues in the reduced form (monoisotopic molecular mass 3161.80 Da), as can be deduced from the lower intensity of the peak at
m⁄ z 3161.83 and the higher intensity of peaks from
m⁄ z 3162.86 to m ⁄ z 3165.79 compared with the theor-etical isotope distribution expected for the peptide with the S–S bridge (Fig 6)
The S–S pattern of the other cysteine residues (136,
162, 190, 202) was determined cleaving the peptide chain between Cys136 and Cys162, by means of tryptic digestion of the protein In the MALDI-TOF mass spectra the signal at m⁄ z 3022.39 could be assigned
to the pairing of the two peptides 158–167 (monoiso-topic molecular mass 1081.48 Da) and 179–197
0
0
0
0
0
1
8 0 6
0 4
0 2
0 0
[ l o t i e h t o i h t i
Fig 5 Effect of reducing agents on PfPNP thermostability The
enzyme (2 lg) was incubated for 60 min in 20 m M Tris ⁄ HCl pH 7.4
containing dithiothreitol at indicated concentrations at 70 C (d),
80 C (j), 90 C (m), and 100 C (s) Aliquots were then withdrawn
and assayed for PNP activity as described under Experimental
procedures.
Trang 8(monoisotopic molecular mass 1942.00 Da) thus
indi-cating that Cys162 is linked to Cys190 Similarly, the
signal at m⁄ z 4371.87 could be generated by the
pep-tides 125–140 (average molecular mass 1923.17 Da)
and 198–220 (average molecular mass 2450.87 Da)
linked by a disulfide bond between Cys136 and Cys202
(Table 3) The S–S arrangement was further confirmed
by submitting the tryptic peptide mixture to tandem
mass spectrometric experiments As an example, the
MS⁄ MS analysis of the peptide containing the S–S
bond between Cys162 and Cys190 is reported in detail
The triply charged ion at m⁄ z 1008.14, generated from
disulfide-containing peptide (158–167) + (179–197),
was selected for CID experiments and Fig 7 reports
the MS⁄ MS spectrum and the peptide amino acid
sequence Fragment ions belonging to series b
(con-taining the N-terminal region of the peptide) and
y (containing the C-terminal region) were originated
from the entire sequence of both peptides 158–167 and 179–197 Diagnostic fragment ions of the S–S pairing resulted to be the singly charged ion y7 (m⁄ z 769.44) originated from the fragment 191–197 and its comple-mentary doubly charged ion b12 (m⁄ z 1127.50) origin-ated from the fragment 179–190 linked to the intact peptide 158–167 This is further demonstrated by the singly charged ion y5 (m⁄ z 559.27) produced from the fragment 163–167 and by the complementary doubly charged ion b5(m⁄ z 1232.51) originated from the frag-ment 158–162 linked to the intact peptide 179–197 It
is interesting to note that the disulfide bonds 136–202 and 254–256 are conserved in PfMTAP and
SsMTAP-II confirming the disulfide arrangement of PfPNP The presence of three disulfide bonds justify the extreme stability features of PfPNP These covalent links, in fact, lowering the entropy of the unfolded poly-peptide and introducing at the same time new molecular
z /
0
0 1
0 1 5 1 0 1
z / m
6 2 1 0 3 1 3 1 1
0 4 1
9 5 1
0 0 1
0 1 0
1 0 0
0 1
n i t u i r s i d e o t o s i a c i t e r o h
1 6 0 0 1
0 1 0 1 1
9 7 1 2 1
6 8 1 3 1
1 8 1 4 1
7 7 1 5 1
n i t u i r s i d e o t o s i a t n m i r e x E
2 3 0 0 1
1 6 3 1 1
0 1 6 2 1
7 9 0 3 1
9 8 0 4 1
2 5 9 5 1
z /
B A
Fig 6 Isotope distribution of the signal at
m ⁄ z 3160.80 originated from the peptide
236–265 with a disulfide bridge
Experimen-tal (A) and theoretical (B) isotope
distribu-tions are shown.
Table 3 Disulfide arrangement of PfPNP The solid lines indicate S–S bridges exactly assigned, while dashed lines refer to S–S bridges which could not be assigned in the experiment.
Experimental m ⁄ z-values Amino acid sequence of disulfide-containing peptides
Disulfide pattern obtained from CNBr reaction
3761.25 231QKKSEDIVKLILAAIPLIPKERRCGCKDALKGATG265
13893.61 92 KPGDFVILDQIIDFTVSRPRTFYDGEESPHERKFVAHVDFTEPY
CPEIRKALITAARNLGLPYHPRGTYVCTEGPRFETAAEIRAYRILGGDVVGM187
188TQCPEAILARELEM201
202
CYATVAIVTNYAAGM216
Disulfide pattern obtained from tryptic digestion
140
GTYVCTEGPR ILGGDVVGMTQCPEAILAR197
4371.87 125FVAHVDFTEPYCPEIR ELEMCYATVAIVTNYAAGMSGKK220
Trang 9interactions into the protein structure could be
respon-sible for increasing the kinetic stability that is in turn
responsible for trapping the protein in its native state
also in the extreme environmental conditions
Characterization of C254S⁄ C256S mutant and role
of the CXC motif
To elucidate if the disulfide CGC localized at the
C-terminus of PfPNP, in spite of its unusual structural
features, could play a role in the stabilization of the
protein we utilized site-directed mutagenesis to
substi-tute Cys254 and Cys256 with serine The large-scale
preparation of the C254S⁄ C256S mutant was
per-formed as described above for recombinant PfPNP
Purified mutant protein showed, under either native
(gel filtration) or denaturing (SDS⁄ PAGE) conditions
Mrvalues identical to the wild-type PfPNP and proved
to be fully active indicating the compatibility of the
substitutions with the native state of the protein We
then carried out the characterization of the thermal
properties of the mutant in comparison with those of
PfPNP The results obtained indicate that the
substitu-tion of Cys254 and Cys256 with serine significantly
affect both thermodynamic stability (Tm, 102C) and
kinetic stability (38% residual activity after 4 h
incuba-tion at 100C, half-life of 35.5 min at 105 C) of the
enzyme suggesting an important role of the pair
Cys254-Cys256 in the thermal stabilization of the
enzyme
Disulfide bonds between cysteine residues separated
by a single amino acid are extremely rare in nature In
addition to the disulfide CGC in PfMTAP [26] and
CSC in SsMTAPII [24], the two highly PfPNP
homol-ogous enzymes, only few examples are present in the literature [39–43] The following considerations allowed
us to hypothesize that the presence of a conserved unusual CXC disulfide in PfPNP, PfMTAP and SsM-TAPII would be not casual Firstly, a CGC motif in a mutant of E coli thioredoxin reductase [43] displays a disulfide reduction potential that is close to that of protein disulfide isomerase This soluble eukaryotic protein is the most efficient known catalyst of the formation and isomerization of disulfide bonds [44], especially those within kinetically trapped, structured folding intermediates [45] Second, a strict analogy may be observed between the CSC motif in SsMTAPII and the CGC motif in the thiol oxidase Erv2p from yeast, a FAD-dependent protein that can promote disulfide bond formation during the protein biosynthe-sis in the yeast endoplasmic reticulum [42] In fact, as demonstrated by the elucidation of the three-dimen-sional structure, either in SsMTAPII [20] or in Erv2p [42] the CXC motif is part of a flexible C-terminal segment that can swing into the vicinity of another cysteine pair In particular, in Erv2p the CGC motif was found to be involved in a disulfide relay that may help to shuttle electrons between dithiols of the sub-strate protein and the FAD-proximal disulfide [42] Third, in analogy with Erv2p, the CGC motif of PfPNP is localized in the C-terminus of the enzyme that, as indicated by the protease sensitivity of the polypeptide chain at neighboring residues, is a flexible region All these considerations and the results indica-ting a reduced thermodynamic and kinetic stability of the mutant C254S⁄ C256S with respect to the wild-type PfPNP, suggest that, as already hypothesized for SsM-TAPII [20,24], the two cysteines of the CGC motif in
Fig 7 MS ⁄ MS spectrum of the peptides 158–167 and 179–197 linked by S–S brid-ges Diagnostic fragment ions b5and y5 originated from the peptide 158–167, while ions b* and y* were from the peptide 179–197.
Trang 10PfPNP can undergo reversible oxidation-reduction to
rescue the possible damage of the other two disulfide
bonds The presence of a low percentage of the protein
with Cys254 and Cys256 in the reduced form further
supports this hypothesis
It has been recently demonstrated that specific
pro-tein disulfide oxidoreductases, structurally and
functio-nally related to eukaryotic protein disulfide isomerase,
play a key role in intracellular disulfide-shuffling in
hyperthermophilic proteins [46–48] In addition to
protein disulfide oxidoreductases, the oxidized CXC
motif in hyperthermophilic enzymes with intrasubunit
disulfide bonds, such as PfPNP, PfMTAP, and
SsM-TAPII, could represent an ingenious strategy adopted
by these proteins to preserve their folded state in the
extreme conditions
Experimental procedures
Bacterial strains, plasmid, enzymes
and chemicals
MTA was prepared from AdoMet [23] Thermolysin and
Endoproteinase Glu-C were obtained from Boehringer
(Mannheim, Germany)
O-Bromoacetyl-N-hydroxysuccini-mide, cytochrome c, trypsin, cyanogen bromide (CNBr),
angiotensin, adrenocorticotropic hormone fragment 18–39;
nucleosides, purine bases and standard proteins used in
(St Louis, MO, USA) Dithiothreitol and
isopropyl-b-d-thiogalactoside were from Applichem (Darmstadt,
Ger-many) Sephacryl S-200 and AH-Sepharose 4B were
obtained from Amersham Pharmacia Biotech;
polyvinyli-dene fluoride membranes (0.45 mm pore size) were obtained
from Millipore (Bedford, MA, USA.) Specifically
synthes-ized oligodeoxyribonucleotides were obtained from
MWG-Biotech (Ebersberg, Germany) Plasmid pET-22b(+) and
the NucleoSpin Plasmid kit for plasmid DNA preparation
were obtained from Genenco (Duren, Germany) E coli
strain BL21(kDE3) was purchased from Novagen
(Darms-tadt, Germany) P furiosus chromosomal DNA was kindly
provided by C Bertoldo (Technical University,
Hamburg-Harburg, Germany) Restriction endonucleases and
DNA-modifying enzymes were obtained from Takara Bio, Inc
(Otsu, Shiga, Japan) Pfu DNA polymerase was purchased
from Stratagene (La Jolla, CA, USA) Nonspecific
adeno-sine deaminase was purified 200-fold from Aspergillus
USA) according to Wolfenden et al [49]
Enzyme assay
Purine nucleoside phosphorylase activity was determined
following the formation of purine base from the
corres-ponding nucleoside by HPLC using a Beckman system Gold apparatus The assay was carried out as already reported [25] Unless otherwise stated, the standard incuba-tion mixture contained the following: 20 lmol potassium phosphate buffer, pH 7.4, 400 nmol of the nucleoside and the enzyme protein in a final volume of 200 lL The incu-bation was performed in sealed glass vials for 5 min at
experi-ments in the absence of the enzyme were performed in order to correct for nucleoside hydrolysis When the assays
mixture was preincubated for 2 min without the enzyme that was added immediately before starting the reaction
An Ultrasphere ODS RP-18 column was employed and the
retent-ion times of inosine and hypoxantine, guanosine and guan-ine were 10.5 min and 4.7 min, and 11.5 min and 4.3 min, respectively The amount of purine base formed is deter-mined by measuring the percentage of the absorbance integrated peak area of purine base formed with respect to the total (nucleoside + purine base) absorbance integrated peak areas In all of the kinetic and purification studies the amounts of the protein was adjusted so that no more than 10% of the substrate was converted to product and the reaction rate was strictly linear as a function of time and protein concentration One unit of enzyme activity was defined as the amount of enzyme that catalyzes the cleavage
Determination of kinetic constants
Homogeneous preparations of PfPNP were used for kinetic studies The purified enzyme gave a linear rate of reaction
5 min was employed for kinetic experiments All enzyme reactions were performed in triplicate Kinetic parameters were determined from Lineweaver–Burk plots of initial
linear regression analysis of data fitted to the Michaelis– Menten equation Values given are the average from at
molecular mass of 180 kDa
Analytical methods for protein
Protein concentration was determined by means of the Bradford method [50] using bovine serum albumin as the standard The molecular mass of the native protein was determined by gel filtration on a calibrated Sephacryl S-200 column as already reported [24] The molecular mass under dissociating conditions was determined by SDS polyacryla-mide gel electrophoresis, as described by Weber et al [51]