Moreover, we determined the pattern of disulfide bridges for the first time and demonstrated, on the basis of equilibrium studies of guanidinium chloride GdmCl-induced denaturation in the
Trang 1Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus
Mechanism of the reaction and assignment of disulfide bonds
Giovanna Cacciapuoti1, Maria Angela Moretti2, Sabrina Forte1, Assunta Brio1, Laura Camardella3,
Vincenzo Zappia1and Marina Porcelli1
1 Dipartimento di Biochimica e Biofisica ‘F Cedrangolo’, Seconda Universita` di Napoli, Naples, Italy; 2 Centro Regionale di Competenza in Biotecnologie Industriali (BioTekNet), Seconda Universita` di Napoli, Naples, Italy; 3 Istituto di Biochimica delle Proteine, CNR, Naples, Italy
The extremely heat-stable 5¢-methylthioadenosine
phos-phorylase from the hyperthermophilic archaeon Pyrococcus
furiosuswas cloned, expressed to high levels in Escherichia
coli, and purified to homogeneity by heat precipitation and
affinity chromatography The recombinant enzyme was
subjected to a kinetic analysis including initial velocity and
product inhibition studies The reaction follows an ordered
Bi–Bi mechanism and phosphate binding precedes
nucleo-side binding in the phosphorolytic direction
5¢-Methyl-thioadenosine phosphorylase from Pyrococcus furiosus is a
hexameric protein with five cysteine residues per subunit
Analysis of the fragments obtained after digestion of the
protein alkylated without previous reduction identified two
intrasubunit disulfide bridges The enzyme is very resistant
to chemical denaturation and the transition midpoint for
guanidinium chloride-induced unfolding was determined to
be 3.0Mafter 22 h incubation This value decreases to 2.0M
in the presence of 30 mMdithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE The finding of multiple disulfide bridges in 5¢-methylthioadenosine phosphorylase from Pyrococcus furiosusargues strongly that disulfide bond formation may
be a significant molecular strategy for stabilizing intra-cellular hyperthermophilic proteins
Keywords: disulfide bonds; hyperthermostability; 5¢-methyl thioadenosine phosphorylase; purine nucleoside phos-phorylase; Pyrococcus furiosus
Hyperthermophilic enzymes which retain their structure
and function near the boiling point of water have been, over
the past decade, the object of extensive studies on protein
stabilization, folding and evolutionary aspects [1–4]
More-over, their unique structure–function properties of high
thermostability are potentially significant for developing
biotechnological applications [5,6] Thus, there is a great
deal of interest in studies on the biochemical adaptation of
hyperthermophiles whose enzymes provide unique models
for the study and understanding of the evolution of enzymes
in terms of structure, specificity and catalytic properties
Much work has been done to identify the structural determinants of the enhanced stability of hyperthermophilic proteins Several mechanisms of thermal stabilization have been proposed, among which additional networks of salt bridges and hydrogen bonds, improved packing density and enhanced secondary structure are the most cited [2–4,7–9]
In spite of this, no general rules have been established to date, and it has been concluded that each protein evolves individually through a limited number of factors that occur
at different levels, also involving the amino acid sequence and the quaternary structure of the proteins
In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hyperthermo-philic proteins where these covalent links may play a key role in protein stabilization in the extreme thermal environ-ment [10–14]
5¢-Methylthioadenosine phosphorylase (MTAP) cata-lyzes the reversible phosphorolysis of 5¢-methylthioadeno-sine (MTA), a sulfur-containing nucleoside formed from S-adenosylmethionine (AdoMet) via several independent pathways of which the polyamine biosynthesis is quantita-tively the most important [15] The products of the MTA cleavage reaction are adenine and 5-methylthioribose-1-phosphate MTA phosphorylase was first characterized
in rat ventral prostate [16] The enzyme was purified to homogeneity from mammalian tissues [17–19] and from the
Correspondence to G Cacciapuoti, Dipartimeno di Biochimica e
Biofisica F Cedrangolo, Seconda Universita` di Napoli, Via
Costantinopoli 16, 80138, Napoli, Italy Fax: +39 081 441 688;
Tel.: +39 081 566 7519; E-mail: giovanna.cacciapuoti@unina2.it
Abbreviations: AdoHcy, S-adenosyl- L -homocysteine; AdoMet,
S-adenosylmethionine; GdmCl, guanidinium chloride; hMTAP,
human MTAP; IPTG, isopropyl thio-b- D -galactoside; MTA,
5¢-methylthioadenosine; MTAP, 5¢-methylthioadenosine
phosphory-lase; PfMTAP, 5¢-methylthioadenosine phosphorylase from
Pyro-coccus furiosus; PNP, purine nucleoside phosphorylase; SsMTAP,
MTAP from Sulfolobus solfataricus; TFA, trifluoroacetic acid.
(Received 26 July 2004, revised 12 October 2004,
accepted 22 October 2004)
Trang 2Archaea Sulfolobus solfataricus [20] and Pyrococcus furiosus
[21] Moreover, crystal structures have been obtained for
human MTAP (hMTAP) [22] and for MTAP from
S solfataricus(SsMTAP) [10]
5¢-Methylthioadenosine phosphorylase from Pyrococcus
furiosus(PfMTAP) is a member of the purine nucleoside
phosphorylase (PNP) family of enzymes, which function in
the purine salvage pathway of cells [23]
PNP are classified into two main categories:
low-molecular mass PNP, homotrimers, specific for catalysis
of 6-oxopurines and their nucleosides [23], and
high-molecular mass PNP, homohexamers, with broad substrate
specificity in that they accept both 6-oxo- and/or
6-amino-purines and their nucleosides as substrates [23] The two
classes do not have sequence homology but the analysis of
the three-dimensional structure of their monomers showed
significant similarity [24,25]
PfMTAP can be considered a PNP with unique features
[21] In fact, because of its hexameric quaternary structure,
this enzyme belongs to the high-molecular mass class of
PNP By contrast, because of its amino acid sequence,
PfMTAP is more similar to hMTAP, a trimeric enzyme
with high substrate specificity for MTA [22]
PfMTAP is highly thermoactive with an optimum
temperature of 125C and is extremely thermostable,
retaining 98% residual activity after 5 h at 100C and
showing a half-life of 43 min at 130C [21] The enzyme
is also extremely stable to proteolytic cleavage and after
incubation with protein denaturants, detergents, organic
solvents, and salts even at high temperature [21] PfMTAP
contains 30 cysteine residues (five per subunit) These
residues, on the basis of biochemical evidence such as
decrease of the thermal stability in the presence of
dithio-threitol and different mobility levels of the enzyme on SDS/
PAGE run under reducing and nonreducing conditions, are
thought to be involved in intrasubunit disulfide bonds [21]
We describe here the in vitro expression, purification and
characterization of the hyperthermostable PfMTAP We
carried out a detailed kinetic investigation in order to clarify
the mechanism of the reaction and the sequence of binding
of substrates Moreover, we determined the pattern of
disulfide bridges for the first time and demonstrated, on the
basis of equilibrium studies of guanidinium chloride
(GdmCl)-induced denaturation in the presence and absence
of reducing agents, that disulfide bonds are needed for
PfMTAP stability
Materials and methods
Bacterial strains, plasmid, enzymes, and chemicals
Plasmid pET-22b(+) and the NucleoSpin Plasmid kit for
plasmid DNA preparation were obtained from Genenco
(Duren, Germany) Escherichia coli strain BL21(kDE3) was
purchased from Novagen (Darmstadt, Germany) P
furio-suschromosomal DNA was kindly provided by C Bertoldo
(Technical University Hamburg-Harburg, Germany)
Specifically synthesized oligodeoxyribonucleotides were
obtained from Primm (Naples, Italy) Restriction
endonuc-leases and DNA-modifying enzymes were obtained from
Takara Bio, Inc (Otsu, Shiga, Japan) Pfu DNA
poly-merase was purchased from Stratagene (La Jolla, CA,
USA) [methyl-14C]AdoMet (50–60 mCiÆmmol)1was sup-plied by the Radiochemical Centre (Amersham Bioscience, Buckinghamshire, UK) MTA and 5¢-[methyl-14C]MTA were prepared from unlabeled and labeled AdoMet [26] and purified by HPLC [27] Sephacryl S-300, AH-Sepharose 4B, S-adenosyl-L-homocysteine (AdoHcy), adenosine, adenine, guanosine, guanine, inosine, hypoxanthine, O-bromoacetyl-N-hydroxysuccinimide and standard proteins used in molecular mass studies were obtained from Sigma (St Louis, MO, USA) GdmCl and dithiothreitol were from Applichem (Darmstadt, Germany) 4-Vinylpyridine and CNBr were purchased from Aldrich (Steinheim, Germany) PD-10 columns were from Amersham Pharmacia Biotech All reagents were of the purest commercial grade
Enzyme assay MTA phosphorylase activity was determined by measuring the formation of [methyl-14 C]5-methylthioribose-1-phos-phate from 5¢-[methyl-14C]MTA [20] Unless otherwise stated, the standard incubation mixture contained the following: 20 lmol potassium phosphate buffer, pH 7.4,
80 nmol of [methyl-14C]MTA (6.5· 105cpmÆlmol)1), and the enzyme protein in a final volume of 200 lL The incubation was performed in sealed glass vials for 5 min at
80C, except where indicated otherwise The vials were rapidly cooled in ice, and the reaction was stopped by the addition of 100 lL of 10% (v/v) trichloroacetic acid The mixture was then applied to a Dowex 50-H+ column (0.6· 2 cm) equilibrated in H2O 5-[methyl-14 C]Methylth-ioribose-1-phosphate produced was eluted with 2.5 mL of 0.01MHCl directly into scintillation vials and counted for radioactivity Control experiments in the absence of the enzyme were performed in order to correct for MTA hydrolysis When the assays were carried out at tempera-tures above 80C, the reaction mixture was preincubated for 2 min without the enzyme, which was added immedi-ately before starting the reaction
When inosine, guanosine, and adenosine were used as substrates, the formation of purine base was measured by HPLC using a Beckman system Gold apparatus The amount of purine base formed is determined by measuring the percentage of the absorbance integrated peak area of purine base formed with respect to the total (nucleo-side + purine base) absorbance integrated peak areas An Ultrasil-CX column (Beckman) eluted with 0.05M ammo-nium formate, pH 3 at a flow rate of 1 mLÆmin)1was used when adenosine and/or guanosine were the substrates of the reaction In these experimental conditions the retention times
of adenosine and adenine, guanosine and guanine were 7.3 and 12.4 min, and 4.2 and 6 min, respectively When the assays were carried out in the presence of inosine as substrate,
an Ultrasphere ODS RP-18 column was employed and the elution was carried out with 5 : 95 (v/v) mixture of 95% methanol and 0.1% trifluoroacetic acid (TFA) in H2O The retention times of inosine and hypoxanthine were 10.5 and 4.7 min, respectively The same HPLC assay has been carried out with unlabeled MTA as substrate In this case an Ultrasphere ODS RP-18 column was equilibrated and eluted with 20 : 80 (v/v) mixture of 95% methanol and 0.1% TFA
in H2O The retention times of MTA and adenine were 10 and 4.2 min, respectively
Trang 3In product inhibition studies, 0.4 lg of enzyme protein in
a final volume of 200 lL were employed The reaction was
carried out in the presence of 2 lmol of potassium
phosphate buffer pH 7.4 when MTA and adenosine were
the variable substrates, and in the presence of 5 lmol Hepes
buffer pH 7.4 when phosphate was the variable substrate
In all of the kinetic and purification studies the amount 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
All enzyme reactions were performed in triplicate at
80C Km and Vmax values were obtained from linear
regression analysis of data fitted to the Michaelis–Menten
equation
Cloning and expression of the PfMTAP-encoding gene
The previously obtained N-terminal amino acid sequence of
PfMTAP [21] was used for BLAST search of the complete
genome sequence of P furiosus (http://comb5-156.umbi
umd.edu/)
The coding region of PfMTAP was cloned into the
pET-22b(+) expression vector via two engineered restriction sites
(NdeI and BamHI) introduced by PCR with the following
primers 5¢-GACGGTGATACATATGCCCAAGATAG
GG-3¢, sense, and 5¢-GCAGCTACAAGGATCCAAAG
TAAATAGG-3¢, antisense (the introduced restriction sites
are underlined) Isolated genomic P furiosus DNA (20 ng),
hydrolyzed using BamHI was used as a template PCR
amplification was performed with P furiosus DNA
polym-erase and a Minicycler (Genenco) programmed for 29
cycles, each cycle consisting of denaturation at 92C for
1 min, annealing at 55C for 2 min and extension at 72 C
for 2 min plus 5 sÆcycle)1, followed by an extension final
step of 15 min at 72C The amplified gene (25 ng),
hydrolyzed using NdeI and BamHI was inserted into
pET22b(+) (150 ng) cut with the same restriction enzymes
The recombinant plasmid was named pET-MTAP The
nucleotide sequence of the inserted gene was determined by
MWG BIOTECH to ensure that no mutations were present
in the gene
For the expression of recombinant PfMTAP, an
over-night culture of E coli BL21 (kDE3) transformed with the
plasmid pET-MTAP was used as 0.5% inoculum in 1 L of
Luria–Bertani medium [28] containing 100 lgÆmL)1
ampi-cillin at 37C At a late stage of cellular growth (when the
culture reached an optical density of 3.0) isopropyl
thio-b-D-galactoside (IPTG) was added to 1 mMfinal
concen-tration and the induction was prolonged for 16 h Cells were
harvested by centrifugation and lysed as described by
Sambrook et al [28] The cell debris was removed by
centrifugation at 20 000 g for 60 min at 4C and the
supernatant was used as a cell-free extract
Purification of recombinant PfMTAP
Recombinant PfMTAP was purified in two steps The
cell-free extract of BL21 E coli cells expressing PfMTAP was
heated at 100C for 10 min and centrifuged at 20 000 g for
60 min After dialysis overnight against 10 mM Tris/HCl
pH 7.4, the enzyme was applied to an affinity column of
AdoHcy Sepharose (2· 12 cm) prepared as described by Porcelli et al [29] equilibrated with 20 mMTris/HCl pH 7.4 The column was washed stepwise with 50 mL of the equilibration buffer and then with the same buffer containing 0.5M NaCl until the absorbance at 280 nm reached the baseline MTA phosphorylase activity was then eluted with
20 mMTris/HCl pH 7.4 containing 0.5MNaCl and 3 mM
MTA Active fractions were pooled, concentrated and dialyzed extensively against 10 mMTris/HCl pH 7.4 Protein analysis
Proteins were assayed by the Bradford method [30] using bovine serum albumin as standard Protein eluting from the columns during purification was monitored as absorbance at 280 nm The concentration of purified PfMTAP was estimated spectrophotometrically using
e280¼ 23 500M )1Æcm)1 The molecular mass of the native protein was determined
by gel filtration and nondenaturating PAGE Gel filtration was performed on a calibrated Sephacryl S-300 column (2.2· 95 cm) equilibrated with 10 mM Tris/HCl pH 7.4 containing 0.3M NaCl at a flow rate of 4 mLÆh)1 The column was calibrated by using standard proteins of known molecular mass Nondenaturating PAGE was carried out at
pH 7.5 as reported by Cacciapuoti et al [31] The gels were either stained with Coomassie Blue or cut into thin slices and assayed for MTA phosphorylase activity by incubating in the assay mixture at 80C for 10 min The subunit molecular mass was determined by SDS/PAGE, as described by Weber
et al [32], using 12 or 15% acrylamide resolving gel and 5% acrylamide stacking gel Samples were heated at 100C for
5 min in 2% SDS, 5% 2-mercaptoethanol and run in comparison with molecular mass standards
Enzyme thermostability was tested by incubating the protein in sealed glass vials at temperatures between 100 and
145C Samples (2 lg) were taken at time intervals and residual activity was determined by the standard assay at
80C Activity values are expressed as a percentage of the zero-time control (100%)
Fluorescence spectroscopy Fluorescence emission spectra of tryptophan 69 and 208
of PfMTAP were used to monitor any changes in the environment of these residues upon the unfolding of the protein Intrinsic fluorescence emission measurements were carried out on a Perkin–Elmer (Norwalk, CT, USA) MMF-44 spectrofluorometer in the range of fluorescence linearity using a 1-cm path length quartz cuvette and 5-nm slit width The absorbance of all solutions was 0.05– 0.10 at the excitation wavelength Fluorescence emission spectra were recorded at 300–450 nm at the controlled temperature of 25C with the excitation wavelength set at
290 nm Experiments were corrected for background signal
Equilibrium experiments on GdmCl-induced unfolding and refolding
For unfolding, PfMTAP (final concentration 0.125 mgÆmL)1) was incubated for 22 h at 25C in GdmCl at
Trang 4various concentrations (0–6M) in 20 mMTris/HCl pH 7.4
in the presence and in the absence of 30 mM
dithiothre-itol Unfolding was probed by recording the intrinsic
fluorescence emission After 22 h, refolding was started by
20-fold dilution of the unfolding mixture in Tris/HCl
20 mM pH 7.4 at 25C The final concentration of
GdmCl in the renaturation mixture was 0.3M, whereas
the protein concentration was about 6 lgÆmL)1 The
refolded enzyme, after extensive dialysis against Tris/HCl
20 mM pH 7.4 until complete removal of GdmCl, was
analyzed by intrinsic fluorescence emission, catalytic
activity measurements under standard conditions, and
SDS/PAGE analysis
Protein fragmentation with CNBr and peptide mapping
Purified recombinant PfMTAP was alkylated with
4-vinyl-pyridine under denaturing conditions with and without
previous reduction by the following procedure The enzyme
(0.4 mg, 13.3 nmoles) was dissolved in denaturing buffer
containing 0.5 M Tris/HCl, pH 7.8, 2 mM EDTA, 6M
GdmCl in the presence and in the absence of dithiothreitol
at a 150-fold molar excess over cysteine residues and the
solution was incubated at 40C under nitrogen for 2 h (this
step was omitted in the sample alkylated without previous
reduction) 4-Vinylpyridine (fivefold molar excess over all
thiol groups) was added to the reduced and nonreduced
samples and the reaction proceeded at room temperature in
the dark under nitrogen for 45 min The resulting alkylated
samples were immediately desalted by gel filtration on
prepacked PD-10 column equilibrated with 0.1% (v/v)
TFA, and dried under vacuum Cleavage at methionyl
residues was achieved by dissolving the samples in GdmCl
6M/HCl 0.2M followed by addition of 160-fold molar
excess (over methionine) of CNBr The samples were kept at
25C in the dark for 24 h and then dried under vacuum
The peptide mixture was separated by reverse-phase HPLC
on a 4.6· 250 mm Vydac C18column using a Beckman
system Gold apparatus The elution was accomplished by a
linear gradient from 5 to 60% in 60 min of solvent B (0.08%
TFA in acetonitrile) in solvent A (0.1% aqueous TFA) at a
flow rate of 1 mLÆmin)1 The eluate was monitored at 220
and 280 nm Individual peptide fractions were manually
collected, dried under vacuum, and sequenced
N-Terminal sequencing and mass spectrometric analysis
Peptides were analyzed by automated Edman degradation
using a protein sequencer model Procise 492 from Applied
Biosystem with in line phenylthiohydantoine analysis
Mass spectrometry analysis of individual peptide fractions
were performed by MALDI-MS mass spectrometry on a
Voyager DE Pro mass spectrometer (Applied Biosystem,
Foster City, CA, USA) operating in positive-ion linear
mode Samples were mixed with saturated solution of
a-cyano-4-hydroxycinnaminic acid (10 mgÆmL)1) in
aceto-nitrile/0.2% TFA 70 : 30 (v/v) and applied to the metallic
sample plate before air-drying Mass calibration was
performed with the ions from ACTH (fragment 18–39) at
246 600 Da and cytochrome c at 618 100 Da (MH2)2+as
internal standard Average mass values were measured in
this analysis
Results and Discussion
Analysis of the gene, cloning, expression and purification From the complete genome sequence of P furiosus the gene PF0016, encoding PfMTAP was identified as a 774-bp fragment that, when translated, contained an N-terminus that matched exactly the one determined from the purified enzyme [21] The structural gene of PfMTAP encodes a protein of 257 residues with a predicted molecular mass
of 29 219 Da, which is in good agreement with that of
30 ± 1 kDa estimated by biochemical analyses for the native enzyme [21]
The coding region starts with an ATG triplet, at the position 14 581 of the P furiosus genome, in agreement with data from protein amino acid sequence determination, which indicates that the N-terminal methionine is not post-translationally removed The first stop codon, TGA, is encountered at the position 15 355 Upstream from the coding region, 13 bp before the starting codon, there is a stretch of purine-rich nucleosides (GACGG) that may function as the ribosome-binding site [33] Putative promo-ter elements, which are in good agreement with the archaeal consensus [33], designated box A and box B, were found close to the transcription start site A hexanucleotide with the sequence TAAATA similar to the box A is located
27 bp upstream from the start codon and resembles the TATA box which is involved in binding the archaeal RNA polymerase [33] A putative box B (ATGC) overlaps the ATG codon Finally, a pyrimidine-rich region (TTT TTTAT), strictly resembling the archaeal terminator signal [33], was localized 2 bp downstream from the translation stop codon All these sequences were identified on the basis
of their similarity with those reported in nearby regions of other genes of proteins isolated from P furiosus [34,35] or from other Archaea [33]
As reported for P furiosus and other Pyrococcus genomes [36] a strong bias against the CG dinucleotide is observed in the gene encoding PfMTAP which is reflected in all codons except one proline and one threonine codon In contrast, the CG dinucleotide-containing codons are fre-quently used in E coli [37] The significance of this bias in
P furiosusand other hyperthermophiles may be that, at the optimal growth temperature approaching 100C, cytosine deamination can occur which causes the formation of uracil
in the DNA The subsequent C to T transition will produce
a damage of protein function
The PCR-amplified fragment of PfMTAP was cloned into pET-22b(+) The sequence of the gene was found to be identical with the published PfMTAP sequence (GenBank identifier PF0016) The recombinant PfMTAP was expressed in soluble form in E coli BL21 cells harboring the pET-MTAP plasmid at 37C in the presence of IPTG The most favorable conditions for the expression of the enzyme were found to be when IPTG was added at a late stage of cellular growth and when the induction was prolonged for 16 h Therefore, these conditions were chosen for the large scale production of recombinant PfMTAP and about 12 g of wet cell paste was obtained from 1 L of culture Recombinant PfMTAP was easily purified to homogen-eity 12.6-fold by two-step purification procedure (Table 1) The first step in the purification of the protein from crude
Trang 5cell lysate was an optimized heat precipitation, made
possible by the extreme thermostability of the enzyme As
shown in Fig 1, which reports the analysis by SDS/PAGE
of recombinant PfMTAP at different stages of purification,
most E coli thermolabile proteins can be denaturated and
precipitated by heating and only minor contaminants of
thermostable recombinant PfMTAP are detectable The
remaining impurities were removed by an affinity
chroma-tography on AdoHcy-Sepharose About 9.2 mg of enzyme
preparation with a 43% yield was easily obtained from 1 L
of culture
IPTG-induced E coli cells transformed with pET-MTAP
produced 1.77 mg of recombinant protein per gram of
cells: thus the expression is about 15-fold higher than for
MTAP from P furiosus [21]
The purified recombinant PfMTAP was biochemically
analyzed with respect to molecular properties and compared
with the native enzyme purified from P furiosus The
apparent molecular masses of the enzyme (180 kDa, as
determined by gel filtration) and its subunit (30 kDa, as
judged by SDS/PAGE) were indistinguishable from those of
the native enzyme from P furiosus When compared with
the native PfMTAP, the recombinant enzyme shows the
same features of thermoactivity (optimum temperature
125C) thermoresistance to reversible denaturation (appar-ent Tm 137C after 10 min preincubation as a function
of temperature) and stability in the presence of protein denaturants, and detergents All these data indicate that
in vitroreconstitution of PfMTAP yielded a recombinant hexameric enzyme with properties identical to those of the native enzyme isolated from P furiosus including proper folding
As observed for native PfMTAP [21], in the recombinant enzyme thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present because incubation with 0.8Mdithiothreitol significantly reduces the thermostability of the enzyme Furthermore, we can hypo-thesize that the disulfide linkage(s) are positioned intrasub-unit because, when subjected to SDS/PAGE, the reduced and nonreduced form of the enzyme migrates as a protein band at 30 kDa, which corresponds to the monomer of the enzyme (data not shown)
It is interesting to note that PfMTAP is one of the few disulfide bonds-containing proteins functionally over-expressed in E coli where the folding of proteins with postbiosynthetic modifications as disulfides, could represent
a limiting step in their production [38]
Mechanism of the reaction The reaction catalyzed by PNP is reversible Thermo-dynamically, the equilibrium of the reaction is shifted in favor of nucleoside synthesis However, under physiological conditions, the reaction proceeds in the phosphorolytic direction owing to the rapid removal and metabolism of the phosphorolysis products, i.e purine bases and pentose-1-phosphate [23] In analogy, PfMTAP is able to catalyze the reverse synthetic reaction The incubation for 5 min at
80C of recombinant PfMTAP in the presence of adenine
or guanine or hypoxanthine and ribose-1-phosphate resul-ted in the synthesis of the corresponding nucleosides Like native PfMTAP [21], the recombinant enzyme is characterized by broad substrate specificity toward purine nucleosides In fact, it shows a similar 10-fold higher affinity for MTA (Km147 lM) and adenosine (Km109 lM) with respect to inosine (Km963 lM) and guanosine (Km916 lM)
As previously demonstrated, the Kcat/Kmvalues for MTA (1.66· 105M )1Æs)1) and adenosine (2.09· 105M )1Æs)1), are
20-fold higher than for inosine (9.74 · 103
M )1Æs)1) and guanosine (7.98· 103
M )1Æs)1), and indicate that 6-amino purine nucleosides are the preferred and probably the physiological substrates of the enzyme [21] The broad substrate specificity of PfMTAP towards purine nucleosides
is of interest for potential biotechnological applications It is well known that nonspecific bacterial phosphorylases are useful tools for enzymatic synthesis of nucleoside analogues with chemotherapeutic activity [39] Moreover, a gene therapy for human tumors profits by the differences in substrate specificity of human and E coli PNPs [40]
In order to define the mechanism of the reaction catalyzed by PfMTAP and the sequence of binding of the substrates, a detailed kinetic investigation has been carried out
The double reciprocal plot of the initial velocities at variable concentrations of phosphate and five fixed
Table 1 Purification of recombinant 5¢-methylthioadenosine
phos-phorylase from P furiosus A typical purification from 12 g of wet cells
is shown Specific activity is expressed as lmol MTA cleaved per min
per mg of protein at 80C.
Sample
Total protein mg
Total activity units
Specific activity unitsÆmg)1
Yield
%
Purifi-cation n-fold Crude extract 268 139.36 0.52 100 –
Heat treatment 27.3 125.58 4.6 90.1 8.85
AdoHcy-Sepharose 9.2 60.44 6.57 43.3 12.63
Fig 1 SDS/PAGE of recombinant PfMTAP at different stages of
purification Lane A, molecular mass markers; lane B, E coli BL-21
transformed with pET-MTAP, crude extract (20 lg); lane C, E coli
BL-21 transformed with pET-MTAP after induction with IPTG,
crude extract (20 lg); lane D, the same sample as lane C heated at
100 C for 10 min and cleared by centrifugation at 20 000 g (10 lg);
lane E, the same sample as lane D after affinity chromatography
(2 lg).
Trang 6concentrations of MTA yielded a series of lines
intersect-ing to the left of the vertical axis (Fig 2A) A similar
pattern was observed when MTA was varied at five fixed
concentrations of inorganic orthophosphate (Fig 2B)
The Km values, graphically extrapolated by replotting
the slopes and the intercepts of the primary
double-reciprocal plots vs the double-reciprocal concentrations of the
nonvariable substrates (insets in Fig 2A,B) are
107 ± 6.4 lMfor MTA and 280 ± 14 lMfor phosphate
The obtained results permit us, according to the
consid-erations of Cleland [41], to rule out a ping-pong
mechanism and are consistent with a sequential
mechan-ism A sequential mechanism has been proposed for PNP
from both high- and low-molecular mass class [23],
whereas no evidence for a ping-pong mechanism has
been reported In addition, the existence of ternary
complexes have been revealed by X-ray studies for E coli
[42], calf spleen [43], and human erythrocyte PNP [44] and
for MTAP from Sulfolobus solfataricus [10]
On the basis of steady-state kinetic data, several different kinetic mechanisms have been identified for PNPs isolated from a variety of tissues and species Although a sequential Bi–Bi mechanism has been proposed most often, there is no consensus on whether it is ordered or random, and on the order of substrate binding and product release [23] Similarly, different kinetic mechanisms have been proposed for MTAP, i.e a random-sequential mechanism has been shown for mammalian enzyme [45] and an ordered-sequential mechanism, with MTA as the first substrate to bind and 5-methylthioribose-1 phosphate as the first prod-uct to leave, has been demonstrated for rat lung MTAP [46] and for Drosophila melanogaster MTAP [47]
To verify whether the reaction catalyzed by PfMTAP in the phosphorolytic direction proceeds via an ordered binding of substrates or via a random mechanism, product inhibition studies have been designed From the secondary plots of slopes and intercepts vs the concentrations of inhibitors, shown as insets of Fig 3, an inhibition constant
Fig 2 Two-substrate steady-state kinetics (A) Plot of the reciprocal of initial velocity (V) vs the reciprocal of phosphate concentration (l M ) at 36.7 l M (r), 72.7 l M (m), 126.7 l M (s), 252.7 l M (h) and 504.7 l M (d) MTA concentration In the insets are reported linear replots of the slopes and of the intercepts of plot (A) vs the reciprocal of concentrations of MTA (B) Plot of the reciprocal of initial velocity (V) vs the reciprocal of MTA concentration (l M ) at 100 l M (j), 250 l M (s), 500 l M (r), 2000 l M (m), and 10 000 l M (d) phosphate concentration In the insets are reported linear replots of the slopes and of the intercepts of plot (B) vs the reciprocal of concentrations of phosphate Purified enzyme (0.125 lg) was employed The values of K m extrapolated from the replots are 107 l M for MTA and 200 l M for phosphate.
Trang 7can be determined from the slope replot (Kis) or the intercept
replot (Kii) and is the horizontal intercept on the plot [41]
When MTA or adenosine was varied at fixed
concen-trations of phosphate, both adenine (Fig 3A) and
ribose-1-phosphate (Fig 3B) products exert a noncompetitive
inhibition in a similar experimental protocol By contrast,
when phosphate was varied with MTA or adenosine as a
fixed substrate, adenine acts as a noncompetitive inhibitor
(Fig 3C), whereas a pattern of competitive inhibition was
observed for ribose-1-phosphate (Fig 3D) suggesting that
phosphate and ribose-1-phosphate compete for the same
site or the same enzyme form These product inhibition
studies are consistent with an ordered Bi–Bi mechanism
[41] in which phosphate is the first substrate to add to the
enzyme and ribose-1-phosphate is the last product to
dissociate from the enzyme surface This mechanism
proposal is strengthened by the protection exerted by
phosphate against thermal inactivation, previously
dem-onstrated for native PfMTAP [21], suggesting that
phos-phate forms a binary complex with the enzyme
Equilibrium studies of GdmCl-induced unfolding and refolding
To analyze quantitatively the stability of PfMTAP and to point out the presence of disulfide bond(s) we performed equilibrium transition studies by incubating the enzyme at increasing GdmCl concentrations (0–6M) in 20 mM Tris/ HCl, pH 7.4 for 22 h at 25C in the presence and in the absence of 30 mMdithiothreitol In Fig 4 are reported the denaturation curves determined by monitoring the shift in fluorescence maximum wavelength upon excitation at
290 nm, where only tryptophanyl residues are specifically excited In the native state, PfMTAP exhibits a fluorescence emission maximum at 330 nm typical of a protein with partially buried tryptophanyl residues The denaturation process induced by 6MGdmCl brought about a large red shift and a decrease in fluorescence intensity Both changes are expected for an increased exposure of the tryptophanyl residues to the more polar aqueous solvent The different values of the maximum fluorescence emission wavelength
Fig 3 Product inhibition studies (A) Plot of the reciprocal of the initial velocity with respect to the reciprocal of MTA concentration in the absence (r) and presence of 20 l M (j) and 50 l M (m) adenine The inhibition constants K is and K ii estimated from the replots of the slopes and the intercepts vs the concentration of the inhibitor, shown in the insets, are 12.5 and 33.2 l M , respectively (B) Plot of the reciprocal of the initial velocity with respect to the reciprocal of adenosine concentration in the absence (r) and in the presence of 50 l M (j) and 200 l M (m) ribose 1-phospate In the insets are shown the replots of the slopes and the intercepts vs the concentration of the inhibitor The calculated inhibition constants K is and K ii are 436.2 and 366.9 l M , respectively (C) Plot of the reciprocal of the initial velocity with respect to the reciprocal of phosphate concentration in the absence (r) and in the presence 50 l M (j) and 100 l M (m) adenine In the insets are shown the replots of the slopes and the intercepts vs the concentration of the inhibitor The calculated K is and K ii are 47.3 and 107.7 l M , respectively (D) Plot of the reciprocal of the initial velocity with respect to the reciprocal of phosphate concentration in the absence (r) and in the presence of 50 l M (j) and 200 l M (m) ribose-1-phospate The inhibition constant (Ki) of ribose-1-phosphate calculated from the replot shown in the inset is 113.5 l M
Trang 8observed at the end of transition, i.e 349.3 and 339.8 nm in
the absence and presence of reducing agents, respectively,
suggest significant modifications of the enzyme due to the
reduction of disulfide bond(s) The observed
GdmCl-induced denaturation curves show a single sigmoidal
transition indicating an apparent two-state transition from
the native to the unfolded state without any detectable
intermediate
The 3M GdmCl value of the midpoint transition
observed under nonreducing conditions indicates that the
enzyme is not only extremely thermostable, but also very
resistant to chemical denaturation The addition of 30 mM
dithiothreitol to a GdmCl-induced denaturation
experi-ment shifted the apparent midpoint of the transition for
GdmCl to 2M indicating a significant decrease of
protein stability in the presence of reducing agents This
result and the already observed loss of activity after
incubation of the enzyme at high temperature in the
presence of 0.8M dithiothreitol offer convincing evidence
of the presence of disulfide(s) bonds and suggest the crucial
role played by these covalent links in the stabilization of
the protein
To examine whether the GdmCl-induced unfolding of
PfMTAP is reversible, the refolding reaction was induced
by 20-fold dilution of the sample Extensive dialysis was
then carried out until the complete removal of the
denaturant The refolding process was monitored by
fluorescence measurements, SDS/PAGE and enzymatic
assays As observed in Fig 5, the intrinsic fluorescence
emission intensity of PfMTAP unfolded in 6MGdmCl in
the absence of reducing agents (curve c) was decreased
about twofold as compared with that of the native enzyme
(curve a), indicating that one or both tryptophanyl
residues environment is structurally perturbed by the
denaturant By contrast, the presence of dithiothreitol
(curve d) induces a further decrease in fluorescence
emission intensity, suggesting that the reduction of
disulfide bond(s) represents the structural modification producing the observed spectral changes The observation that after the complete removal of the denaturant and of the reducing agent (curve b) the protein exhibits a fluorescence spectrum with the same features of the native enzyme, i.e a fluorescence maximum centered at 330 nm and a similar value of relative fluorescence intensity, indicates that the denaturation process is reversible Only the denaturation of the enzyme carried out in the presence
of reducing agents proved to be reversible indicating that the presence of intact disulfide bonds interferes with the refolding process of PfMTAP
Owing to the stability of PfMTAP towards 2% SDS at room temperature [21], it has been possible to monitor the native hexameric structure recovery of the protein by SDS/ PAGE The inset in Fig 5 compares the SDS/PAGE pattern of the native and refolded enzyme The samples were subjected to SDS/PAGE without boiling and under nonreducing conditions to obtain a picture of the protein species present After refolding (lane 2), the enzyme migrates as a single band at 180 kDa, corresponding to the molecular mass of the hexameric PfMTAP Further-more, when assayed for catalytic activity, the refolded enzyme shows the same specific activity of the native form
On the basis of the reported data we concluded that PfMTAP represents a rare example, if not the only, reported in the literature so far, of a oligomeric hyperther-mophilic protein with disulfide bonds able to undergo a reversible unfolding Studies are in progress to quantita-tively evaluate the equilibrium and kinetic stability of PfMTAP and to determine the main thermodynamic parameters of the protein
Fig 5 Fluorescence emission spectra of PfMTAP The fluorescence emission spectra were recorded after 22 h incubation at 25 C (A) PfMTAP in 20 m M Tris/HCl, pH 7.4; (B) refolded PfMTAP after unfolding in the presence of 30 m M dithiothreitol; (C) PfMTAP in 6 M
GdmCl in the absence of reducing agents; (D) PfMTAP in 6 M GdmCl
in the presence of 30 m M dithiothreitol The inset shows the SDS/ PAGE pattern of PfMTAP and refolded PfMTAP The samples were subjected to SDS/PAGE without boiling and under nonreducing conditions Lane 1, molecular mass markers; lane 2, PfMTAP (5 lg); lane 3, refolded PfMTAP (5 lg).
Fig 4 GdmCl-induced fluorescence changes of PfMTAP
Fluores-cence changes are reported as k max by monitoring the shift in
fluor-escence maximum wavelength, in 20 m M Tris/HCl, pH 7.4 in the
presence (m) and absence (r) of 30 m M dithiothreitol The spectra
were recorded at 25 C after 22 h incubation.
Trang 9Assignment of disulfide bridges
In order to determine the arrangement of disulfide bridges
in PfMTAP, the protein was alkylated with
4-vinylpyri-dine under denaturing conditions, with and without
previous reduction with dithiotreitol, and then subjected
to CNBr cleavage This acidic cleavage was chosen in
order to minimize disulfide interchange, which could
occur at alkaline pH Figure 6 reports the amino acid
sequence of PfMTAP and shows the position of cysteine
residues and the CNBr peptides The peptide elution
patterns are shown in Fig 7 Peaks corresponding to CB5
(with Cys195) and CB2 (with Cys130 and Cys156) present
in the protein alkylated after reduction (A), are completely
absent in the nonreduced protein (B), whereas the peak
corresponding to CB6 (with Cys246 and Cys248) is still
present although in lower amount In the reduced sample
(A), the large peak CB1 contained the N-terminal peptide,
which does not contain Cys residues In the nonreduced
sample (B), peptides CB2, CB5, and CB6 were found in the same peak, coeluting with the large N-terminal peptide The amino acid sequence indicated that CB2 and CB5 were present in equimolar amounts, and that Cys130 and Cys195 were not alkylated This result suggested that the two peptides are connected by a disulfide bridge between Cys130 and Cys195 Peptide CB6 containing Cys246 and Cys248 was also found in the late large peak, but not in stoichiometric amounts with the previous peptides This finding was most probably due to
a carry over by the other large size peptides Under nonreducing conditions (B) CB6 was found at the same position as in the digestion of the reduced protein (A)
To investigate the oxidation state of the two cysteine residues in positions 246 and 248, CB6 was analyzed by MALDI-TOF/MS Following direct alkylation, the molecular mass of the peptide did not reveal the incorpor-ation of vinylpiridine residues (3777.17 Da), whereas, when the peptide was alkylated after the reducing step, the molecular mass of the peptide increased by 212.2 to 3989.11 Da, indicating the addition of two vinylpiridine groups and demonstrating that Cys246 and Cys248 present
in the peptide are involved in a disulfide bridge It has to be noted that these two cysteines are separated by a single amino acid residue, however, this residue is a glycine which, due to its small size and its conformational freedom, could allow the formation of a disulfide bridge between the two adjacent cysteines
Oxidized CXC sequences are rare in nature The few examples reported in the literature include CSC in Mengo virus coat protein [48], CDC in Bacillus Ak.1 protease [49] and CTC in chaperone Hsp33 from E coli [50] More recently it has been reported that a disulfide with the same CGC sequence found in PfMTAP is present near the C-terminus of a yeast thiol oxidase, and it has been postulated that the enzyme could take advantage of a relatively strained CXC disulfide to perform efficient oxidation [51] It has to be pointed out that in all the listed CXC disulfides, the X residue is small and therefore, the Gly residue found in PfMTAP certainly fits this pattern One may ask what is the structural function in PfMTAP
of the disulfide bridge Cys246–Cys248 that links two cysteine residues so near each other in the sequence? The observations that (a) this disulfide is localized in the C-terminal region of PfMTAP; (b) the C-terminal as well
as the N-terminal region of mesophilic proteins are usually highly flexible and disordered and are thought to be the first
Fig 6 Amino acid sequence alignment of PfMTAP and hMTAP Asterisks indicate the cysteine residues Lines above the sequence indicate the expected CNBr peptides.
Fig 7 HPLC of peptides derived from CNBr cleavage of PfMTAP
alkylated with (A) and without (B) previous reduction with dithiothreitol.
CNBr peptides were identified by amino acid sequencing and discussed
in the text.
Trang 10portion of the protein which undergoes denaturation at high
temperature [52]; and (c) two recently discovered
hypothet-ical MTAPs from the hyperthermophilic Archaea S
solfa-taricus(SS02343) and P furiosus (PF0853) contain cysteine
residues localized at the same positions of PfMTAP, suggest
the hypothesis that a disulfide bond in the C-terminus of
PfMTAP might increase the stability of this protein region
It is also interesting to note that the five cysteine residues of
PfMTAP are well conserved in both hypothetical MTAPs,
suggesting that a similar disulfide bridge pattern may be
present in these proteins and that these covalent links could
represent the molecular strategy of thermal stabilization
adopted by MTAP from hyperthermophilic sources
The high sequence identity (52%) between human and
P furiosusMTAP (Fig 6) allowed us to make a sequence–
structure mapping of the P furiosus enzyme utilizing as a
template the known three-dimensional structure of the
human enzyme [44] The obtained data by Swiss Pdb Viewer
(SPDBD 37B 2000 program) give a good support to the
validity of our experimental results showing that, in the
modeled PfMTAP structure, only the pairs Cys130-Cys195
and Cys246-Cys248 are at a distance compatible with a
disulfide bond (Ca atoms) (5.88 A˚ and 5.65 A˚, respectively),
while Cys156 is too far from all other cysteines to form this
type of link Moreover, in the modeled structure, the
disulfide Cys13-Cys195 appears buried
Disulfide bonds are a typical feature of secretory proteins
and are considered to contribute significantly to their overall
stability [53] In contrast, in intracellular proteins from
well-known organisms, because of the reductive chemical
environment inside the cells [54], the presence of these
covalent links is limited to proteins involved in the
mechanism of response to redox stress [55] or to proteins
catalyzing oxidation–reduction processes [56] The
availab-ility of many completely sequenced hyperthermophilic
genomes has indicated that cysteine residues, in spite of
their high sensitivity to oxidation at high temperature [57],
are present in remarkable amounts in hyperthermophilic
proteins In these proteins, these thermolabile residues are
probably protected against thermal inactivation by being
buried in the protein interior or by their involvement in
specific stabilizing interactions such as metal liganding or
disulfide bridges [4] The presence of disulfide bonds within
several archaeal and thermophilic genomes has been
postulated, taking into account the results of a recent
computational study based on the combination of genomic
data with protein structure [13] Moreover, the increasing
number of solved crystallographic structures has highlighted
the presence of disulfide bonds in several hyperthermophilic
proteins [10–12,14] The results reported here on multiple
disulfide bonds in PfMTAP add a new example, to the few
present in the literature, of an intracellular
hyperthermo-philic protein with disulfide bonds and argue strongly that
intracellular disulfides could represent a significant
mech-anism to achieve superior levels of thermostability
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