In order to get insight into the physiological role of SsMTAPII a comparative kinetic ana-lysis with the homologous 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfatar
Trang 1phosphorylase from the archaeon Sulfolobus solfataricus Giovanna Cacciapuoti1, Sabrina Forte1, Maria Angela Moretti2, Assunta Brio1, Vincenzo Zappia1 and Marina Porcelli1
1 Dipartimento di Biochimica e Biofisica ‘F Cedrangolo’, Seconda Universita` di Napoli, Italy
2 Centro Regionale di Competenza in Biotecnologie Industriali (BioTekNet), Seconda Universita` di Napoli, Italy
5¢-Deoxy-5¢-methylthioadenosine phosphorylase
(MTAP) (EC 2.4.2.28) catalyzes the reversible
phos-phorolysis to free adenine and
5-methylthioribose-1-phosphate [1] of 5¢-deoxy-5¢-methylthioadenosine
(MTA), a sulfur-containing nucleoside generated from
S-adenosylmethionine In eukaryotes, polyamine bio-synthesis represents the major pathway of MTA formation: two moles of MTA are released per mole
of spermine and one mole of MTA per mole of sper-midine [2,3] A phosphorolytic breakdown of the
Keywords
5’-deoxy-5’-methylthioadenosine
phosphorylase; disulfide bonds;
hyperthermostability; purine nucleoside
phosphorylase; Sulfolobus solfataricus
Correspondence
G Cacciapuoti, Dipartimento di Biochimica e
Biofisica ‘F Cedrangolo’, Seconda Universita`
di Napoli, Via Costantinopoli 16, 80138,
Napoli, Italy
Fax: +39 081 5667519
Tel: +39 081 5667519
E-mail: giovanna.cacciapuoti@unina2.it
(Received 14 January 2005, revised 16
February 2005, accepted 17 February 2005)
doi:10.1111/j.1742-4658.2005.04619.x
We report herein the first molecular characterization of 5¢-deoxy-5¢-methyl-thio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21 Purified recombinant SsMTAPII is a homohexamer of 180 kDa with
an extremely low Km (0.7 lm) for 5¢-deoxy-5¢-methylthioadenosine The enzyme is highly thermophilic with an optimum temperature of 120C and extremely thermostable with an apparent Tm of 112C that increases in the presence of substrates The enzyme is characterized by high kinetic sta-bility and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site More-over, the binding of 5¢-deoxy-5¢-methylthioadenosine induces a conforma-tional transition that protected the enzyme against protease inactivation
By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S⁄ C261S and C262S show thermophilicity and thermostability fea-tures significantly lower than those of the wild-type enzyme In order to get insight into the physiological role of SsMTAPII a comparative kinetic ana-lysis with the homologous 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out Finally, the align-ment of the protein sequence of SsMTAPII with those of SsMTAP and human 5¢-deoxy-5¢-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate
Abbreviations
hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; IPTG, isopropyl-b-D-thiogalactoside; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus.
Trang 2thioether is operative in Eukarya [1–4] and Archaea
[5,6] while an hydrolytic cleavage of the molecule
occurs in Bacteria [7] and plants [8]
MTA phosphorylase is a member of purine nucleoside
phosporylases (PNPs), ubiquitous enzymes of purine
metabolism that function in the salvage pathway [9]
PNPs belong to the recently defined nucleoside
phos-phorylase-1 (NP-1) family [10] This family includes
enzymes with a single-domain subunit, The NP-1
family is further divided into two subfamilies on the
basis of amino acid sequence homology, quaternary
structure organization, and substrate specificity [10]
The homotrimeric PNPs, isolated from many
mamma-lian tissues, are specific for the catalysis of 6-oxopurines
and their nucleosides [9], while the homohexameric
PNPs are the dominant form in Bacteria [9] and
Arch-aea [5,6] and are characterized by a broad substrate
specificity They, in fact, accept both 6-oxo- and⁄ or
6-aminopurines and their nucleosides as substrates The
two classes do not have sequence homology but the
analysis of their three-dimensional structures showed
significant similarity of their monomers [9,10] To the
second family of nucleoside phosphorylases (nucleoside
phosphorylase-II) belong enzymes which share a
common two-domain subunit fold and a dimeric
qua-ternary structure and are specific for pirimidine
nucleo-sides [10]
MTA phosphorylase was first characterized in rat
ventral prostate [1] The enzyme was purified to
homo-geneity from mammalian tissues [4,11,12] and from
Sulfolobus solfataricus [5] and Pyrococcus furiosus [6]
Moreover, crystal structures have been obtained for
human MTAP (hMTAP) [13] and for MTAP from
S solfataricus(SsMTAP) [14]
SsMTAP is a hexameric protein of 160 kDa made
up of six identical subunits of 26.5 kDa This enzyme
shows a significant sequence identity to E coli PNP
Like E coli PNP [15], SsMTAP is able to cleave
ino-sine, guanosine and adenosine; unique to SsMTAP,
however, is the ability to recognize MTA as substrate
[5] On the other hand, like SsMTAP,
5¢-deoxy-5¢-methylthioadenosine phosphorylase from P furiosus
(PfMTAP) has a hexameric quaternary structure and a
broad substrate specificity with 20-fold higher catalytic
efficiency for MTA and adenosine than for inosine
and guanosine [6] Nevertheless, PfMTAP shows
negli-gible sequence homology with SsMTAP while it shares
about 50% identity with hMTAP, a trimeric enzyme
with high substrate specificity for MTA [13]
SsMTAP and PfMTAP are both characterized by
exceptionally high thermoactivity and thermostability
with temperature optima and apparent melting
tem-peratures above the boiling point of water Three
intersubunit disulfide bonds have been evidenced in the three-dimensional structure of SsMTAP [14] and two intrasubunit disulfides have been identified in PfMTAP [16] Moreover, it has been demonstrated that these covalent links represent an important structural mech-anism adopted by these enzymes to reach superior levels
of stability SsMTAP and PfMTAP represent two of the very few intracellular proteins with disulfide bridges reported so far in the literature It is well known, in fact, that disulfide bonds are a typical feature of secretory proteins [17] and because of the reductive chemical envi-ronment inside the cells [18] the presence of these cova-lent links in intracellular proteins from well known organisms, is limited to proteins involved in the mechanism of response to redox stress [19] or to pro-teins catalyzing oxidation–reduction processes [20] Analysis of the complete genome sequence of S sol-fataricus, a hypothetical 5¢-deoxy-5¢-methylthioadeno-sine phosphorylase, indicated as MTAPII, has been shown beside the well known SsMTAP The observa-tion that this putative MTAP shares 50% identity with human MTAP allows us to hypothesize that MTAPII
is a completely different enzyme from SsMTAP
To obtain structural information on MTAPII and to study the functional role played by this enzyme in the purine nucleoside phosphorylase pathway of S solfa-taricus, we carried out the expression of the gene and the purification and the physicochemical characteri-zation of this novel MTAP from S solfataricus (SsMTAPII) Moreover, to investigate on the presence
of disulfide bonds and to obtain information on the role played by these covalent links in the enzyme ther-mostability we constructed by site-directed mutagenesis
a single (Cys262Ser) and a double (Cys259Ser-Cys261-Ser) mutant in the C-terminal region of SsMTAPII These mutants were expressed in E coli and purified, and their activities and thermal properties were com-pared with those of the wild-type enzyme Finally, from the comparative kinetic analysis we demonstrate that SsMTAPII is the first MTA-specific MTA phos-phorylase isolated so far from Archaea
Results and Discussion
Cloning, primary sequence comparison, and expression
The analysis of the complete sequenced genome of
S solfataricus revealed an open reading frame (SS02344) encoding a 270 amino acid protein homo-logous to human MTAP which is annotated as SsMTAPII The putative molecular mass of the pro-tein predicted from the gene was 30.14 kDa and the
Trang 3estimated isoelectric point was 6.54 To overproduce
SsMTAPII the gene was amplified by PCR, as
des-cribed in Experimental procedures and cloned into
pET-22b(+) under the T7RNA polymerase promoter
Comparison of the deduced primary structure of
SsMTAPII with enzymes present in GenBank Data
Base indicated that the highest identity was with the
hypothetical MTA phosphorylases from Sulfolobus
tokodaii (77%), A pernix (69%), Pyrococcus horikoshi
(63%) and Pyrococcus abissi (62%) A high identity
was found with the hypothetical MTAPII from
P furiosus(48%) and with the hypothetical PNP from
Aquifex aeolicus (40%) Among the related enzymes
isolated from various sources, SsMTAPII shows high
sequence identity with PfMTAP (63%) and with
hMTAP (51%) while no significant similarity was
found with SsMTAP, the MTAP isolated from S
sol-fataricus[5]
As deduced from the gene, SsMTAPII contains
seven cysteine residues per subunit This evidence
con-firms the current opinion that, in spite of their high
sensitivity to the oxidation at high temperature [21],
this residue is present in remarkable amounts in
hyper-thermophilic proteins where it is probably involved in
stabilizing disulfide bridges [14,16,22–25]
The recombinant SsMTAPII was expressed in
sol-uble form in E coli BL21 cells harboring the plasmid
pET-SsMTAPII at 37C in the presence of IPTG
Under the experimental conditions selected for the
expression of the enzyme, about 12 g of wet cell paste
was obtained from 1 liter of culture SDS⁄ PAGE
ana-lysis of cell-free extract of induced cells revealed an
additional band of approximately 30 kDa which
cor-responded with the calculated molecular mass of the
gene product This band was absent in extracts of
E coli BL21 carrying the plasmid without the insert
The level of SsMTAPII production in E coli BL21
cells harboring pET-SsMTAPII, was found to be of
0.52 unitsÆmg)1at 70 C, confirming that the SsmtapII
gene had been cloned and expressed
Enzyme purification and properties
SsMTAPII has been produced in a soluble form with
levels of up to 8% of the total cell protein
Recombin-ant protein was easily purified to homogeneity
12.5-fold by a two-step purification procedure The soluble
fraction of the cell extract was heated at 100C for
10 min to eliminate considerable amounts of
heat-labile host proteins The remaining impurities were
removed by an affinity chromatography on
MTA-Sepharose About 14 mg of enzyme preparation with a
57% yield was easily obtained from 1 L of culture No
processing occurred at the enzyme’s N-terminus in the
E coli system, as proven by sequence determination of the first 10 amino acid of SsMTAPII
In analogy with SsMTAP, thiol groups are not involved in the catalytic process, as SsMTAPII activity
is not affected by alkylating, mercaptide-forming or oxidizing thiol reagents It is interesting to note, in this respect, that human MTAP has an absolute require-ment for thiol-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds [4] The purified enzyme was found to be homogeneous SDS⁄ PAGE of the final preparation revealed a single band with a molecular mass of 30 ± 1 kDa, which is consistent with the expected mass deduced from the primary amino acid sequence of the enzyme Gel filtra-tion on an analytical column of Sephacryl S-200 sug-gests that, under native conditions, SsMTAPII forms
a hexameric structure with a molecular mass of
180 ± 9 kDa On the basis of these results SsMTAPII, like the homologous MTA phosphorylases purified from S solfataricus and P furiosus, belongs to the hexameric group of the NP-1 family even if, because
of the high amino acid sequence identity, it is more similar to hMTAP, a typical member of homotrimeric PNPs
Thermophilicity, stability, thermostability and substrate protection
The temperature dependence of SsMTAPII activity assayed in the range from 30 to 140C is reported in Fig 1 The enzyme appears highly thermophilic; its
Fig 1 The effect of temperature on SsMTAPII 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 degrees Kelvin.
Trang 4activity increased sharply up to the optimal
tempera-ture of 120C and a 50% activity was still observable
at 133C The Arrhenius plot shows a discontinuity at
about 97C, with two different activation energies
suggesting conformational changes in the protein
struc-ture around this temperastruc-ture
The stability of SsMTAPII to reversible
denatura-tion was investigated by carrying out short time
kinet-ics of thermal denaturation The diagram of the
residual activity after 5 min of preincubation as a
func-tion of temperature, reported in Fig 2A is
character-ized by a sharp transition From the corresponding
plot (see inset) it is possible to calculate a transition
temperature (apparent Tm) of 112C To evaluate the
possible stabilizing effect of substrates on the
thermo-stability of SsMTAPII we measured the melting
tem-perature of the enzyme in the presence of 100 mm
phosphate or 5 mm MTA As shown in Fig 2A, both
molecules exert a similar protection toward
tempera-ture inactivation of the enzyme causing an increase of
the apparent Tm to 119 and 117C, respectively (see
inset) This result indicates that the binding of these
substrate raises the noteworthy conformational
stabil-ity of the enzyme thus reducing its susceptibilstabil-ity to
thermal denaturation A similar substrate protection
against thermal inactivation was observed for
SsMTAP [5,26], PfMTAP [6] and for MTAP from
human placenta [4]
To study the thermostability properties in terms of
resistance to irreversible thermal inactivation, the
residual activity of SsMTAPII after preincubation
at temperatures between 80 and 115C was followed
for up to 4 h (Fig 2B) Thermal inactivation obeyed
first order kinetics at all the temperatures tested
SsMTAPII displayed high thermostability with half-life
of 22 min and 84 min at 115 and 105C, respectively
The activation energy (Ea) for SsMTAPII inactivation
calculated from the Arrhenius plot (inset) was
7055 kJÆmol)1, about sevenfold higher than the
activa-tion energy calculated for the catalyzed reacactiva-tion,
indi-cating a notably high kinetic stability
Kinetic stability has been reported as a feature of
some naturally occurring proteins that are trapped in
their rigid native conformations by an energy barrier
and therefore are resistant to unfolding It has also
been proposed that the resistance to SDS-induced
denaturation is a common property of kinetically stable
proteins and that it represents a probe for identifying
this type of proteins [27] Therefore, we verified this
correlation in SsMTAPII SsMTAPII resulted highly
resistant to SDS-induced denaturation remaining
com-pletely active up to 2% SDS concentration at room
temperature, whereas it is significantly inactivated at
0 20 40 60 80
100
A
B
80 90 100 110 120 130
Temperature (°C)
-8 -7 -6
250 260 270 1/T x10 5
Tm 112°C
-9 -8 -7
250 255 260 265 1/T x10 5
Tm 119°C
-8 -7 -6
250 255 260 265 1/T x10 5
Tm 117 ° C
1 1.5 2
Time (min)
-9 -8
250 260 270 280 1/T x10 5
Fig 2 Thermostability of SsMTAPII (A) Residual SsMTAPII activity after 5 min of incubation at temperatures shown in the absence (s) or in the presence of 100 mM phosphate ( ) or 5 mM MTA (m) Apparent T m values are reported in the inset (B) Kinetics of ther-mal inactivation of SsMTAPII as a function of incubation time The enzyme was incubated at 80C (d), 90 C (4), 100 C ( ), 105 C
(m) and 115 C (s) for the time indicated Aliquots were then with-drawn and assayed for the activity as described under Experimental procedures The derived Arrhenius plot is reported in the inset.
Trang 570C (Fig 3A) The remarkable SDS-resistance of
SsMTAPII suggests a structural rigidity of the protein
and the occurrence of reduced local and
global-unfold-ing transitions Figure 3A also shows the protective
effect exerted by 100 mm phosphate or 5 mm MTA on the stability of SsMTAPII in the presence of 2% SDS
at 70C The binding of MTA is able to completely stabilize the enzyme that, after a 1-h incubation, remains fully active On the other hand, a lower protec-tion (55% residual activity) is observed in the presence
of phosphate This protective effect indicates that both MTA and phosphate are able to form binary complexes with the enzyme suggesting the hypothesis that, in anal-ogy with human MTAP [28] also SsMTAPII could act via a random mechanism On the contrary, very recently it has been demonstrated that the reaction catalyzed by MTAP from P furiosus follows an ordered Bi-Bi mechanism with the phosphate binding preceding the nucleoside binding in the phosphorolytic direction [16] On the basis of two kinds of evidence a similar ordered Bi-Bi mechanism could also be pro-posed for MTAP from S solfataricus In fact, crystal-lization experiments showed that this enzyme forms a binary complex with phosphate while MTA form only
a ternary complex in cocrystallization with the enzyme and sulfate [14] Furthermore, substrate-stabilization experiments showed that phosphate and ribose-1-phos-phate are able to protect SsMTAP against denaturation
by SDS while MTA and adenine are not [26] To fur-ther analyze the stability of SsMTAPII we carried out equilibrium transition studies by incubating the enzyme
at increasing guanidinium chloride concentrations in
20 mm Tris HCl, pH 7.4 for 22 h at 25C Fig 3B shows the denaturation curve determined by monitor-ing the changes in fluorescence maximum wavelength upon excitation at 290 nm where only tryptophanyl residues are specifically excited [29] The obtained de-naturation curve shows a single sigmoidal transition indicating an apparent two-state transition from the native to the unfolded state without any detectable intermediate The 3.3 m guanidinium chloride value of the midpoint transition indicates that SsMTAPII is very resistant to chemical denaturation
To examine whether the guanidinium chloride-induced unfolding of SsMTAPII 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 guanidinium chloride-induced unfolding proved to be reversible since the refolded enzyme, when assayed for catalytic activity, shows the same specific activity of the native form
Substrate-induced conformational changes The features of unusual stability of SsMTAPII against thermal inactivation and its notably high resistance to
Fig 3 (A) Effect of MTA and phosphate on the thermostability of
SsMTAPII in the presence of 2% SDS The enzyme was incubated
at 70 C with 2% SDS in the absence (s) and in the presence of
100 mM phosphate ( ) or 5 mM MTA (m) At the time indicated,
aliquots were withdrawn and assayed for MTA phosphorylase
activ-ity as described under Experimental procedures Activactiv-ity values are
expressed as percentage of the time-zero control (100%) (B)
guan-idinium chloride-induced fluorescence changes in SsMTAPII
Fluor-escence changes are reported as kmaxby monitoring the shift in
fluorescence maximum wavelength, in 20 mM Tris ⁄ HCl, pH 7.4.
The spectrum was recorded at 25 C after 22-h incubation.
Trang 6SDS- and guanidinium chloride-induced denaturation
are indicative of a compact and rigid structure that
allows protein to retain enzymatic function in the
extreme experimental conditions It has been reported
that, in addition to SDS resistance, kinetic stability is
correlated with resistance to proteolytic cleavage [27]
To verify this correlation in SsMTAPII and to obtain
information about the flexible regions of the protein
exposed to the solvent and susceptible to proteolytic
attack, we subjected the enzyme to limited proteolysis
This technique is also useful for probing
conformat-ional changes occurring in proteins after enzyme–
substrate interaction
Recombinant SsMTAPII was subjected to
proteo-lysis with three different proteases Among these,
trypsin was not able to cleave the enzyme while
sub-tilisin and proteinase K produced essentially the same
results, so only the results for one, proteinase K, are
shown (Fig 4) The time course for the hydrolysis of
recombinant SsMTAPII with proteinase K (Fig 4A)
followed by SDS⁄ PAGE (Fig 4B) showed that a
protein band with an apparent molecular mass of
about 4.5 kDa less than that of SsMTAPII appears
as the catalytic activity decreases After 2-h
incuba-tion the protein band becomes abundant (lane 4,
Fig 4B) and the activity drops to 25% (Fig 4A)
The proteolytic fragment was analyzed by Edman
degradation The analysis showed that the
N-termi-nus was preserved, thus indicating that the
proteo-lytic cleavage site is localized in the C-terminal
region These results indicate that, in analogy with
human MTAP [13], the C-terminal peptide of
Ss-MTAPII is necessary for the integrity of the active
site When the experiment was carried out in the
presence of 5 mm MTA, the enzyme remained
com-pletely active (Fig 4A) and the proteolytic process
did not occur (lane 6, Fig 4B)
A change in protein conformation can mask or
uncover a cleavage site, and an alteration in the
lim-ited proteolysis pattern of the protein can be indicative
of conformational changes [30] Thus, the change in
the digestion pattern of SsMTAPII in the presence of
MTA implies that this substrate binds to protein
spe-cifically and induces a conformational change This
protection against proteolysis provides the first direct
evidence of a MTA-induced conformational change in
SsMTAPII
When SsMTAPII was subjected to limited
proteo-lysis in the presence of 100 mm phosphate only a
slight protection on the catalytic activity was
observed at the early stage of incubation (Fig 4A)
Furthermore, the proteolytic pattern after 2-h
incuba-tion remains almost unmodified (lane 5, Fig 4B)
indicating that phosphate does not bind SsMTAPII
in its C-terminal region
The conclusions drawn from limited proteolysis experiments are strengthened by the analysis of the sequence alignment of SsMTAPII and hMTAP repor-ted in Fig 5 that clearly shows that the C-terminal region of SsMTAPII contains, in well conserved posi-tions, the same residues that in hMTAP are involved
in the binding with MTA [13] with the exception of Leu237 of hMTAP that is substituted in SsMTAPII with Thr229
0 20 40 60 80
100
A
B
Time (min)
50 40
20 25 30
15
Fig 4 Limited proteolysis of SsMTAPII with proteinase K and sub-strate protection (A) Time course for the hydrolysis of SsMTAPII in the absence (s) and in the presence of 100 mM phosphate ( ) or
5 mM MTA (m) (B) SDS PAGE, lane M, molecular mass markers; lane C, SsMTAPII control; lanes 1–4, SsMTAPII (2 lg) after 15-, 30-, 60-, and 120-min incubation in the absence of substrates; lane
5, SsMTAPII after 2-h incubation in the presence of 100 mM phos-phate; lane 6, SsMTAPII after 2-h incubation in the presence of
5 mM MTA The final mass ratio of SsMTAPII to protease was
25 : 1 Aliquots of SsMTAPII at different incubation times at 37 C with protease were taken from the reaction mixture and the hydro-lysis was stopped (see Experimental procedures) Samples were assayed for MTA phosphorylase activity at 70 C, subjected to SDS ⁄ PAGE and gel stained with Coomassie brilliant blue.
Trang 7Effect of reducing agents on enzyme stability
The extreme resistance against the inactivation caused
by temperature, SDS and guanidinium chloride and
the occurrence of a so elevated number of cysteines, 42
in the overall hexamer, prompted us to hypothesize
that SsMTAPII, in analogy with the homologous
MTAP from S solfataricus and P furiosus contains
disulfide bonds This hypothesis is supported by the
observation that five of seven cysteine residues per
SsMTAPII subunit are well conserved in PfMTAP,
where they are involved in these covalent links [6] To
test this hypothesis, we studied the effect of increasing
concentrations of the disulfide-reducing agent
dithio-threitol on SsMTAPII The enzyme is fully stable after
preincubation in the presence of dithiothreitol at
extre-mely high concentrations (0.2, 0.4 and 0.8 m) up to
50C Therefore we have performed thermostability
studies in the presence of these levels of reducing
agents at 80C As shown in Fig 6, at 80 C the
enzyme remains stable up to 0.4 m dithiothreitol
whereas it becomes susceptible to the effect of the
reducing agent as the concentration rises At 0.8 m
dithiothreitol, in fact, a remarkable loss of activity
(30% residual activity) is observable after one hour
incubation This result offers convincing evidence of
the presence of disulfide bonds and suggests a role
played by these covalent links in the stabilization of the protein The requirement of elevated tempera-tures and high concentrations of reducing agents to
Fig 5 Multiple sequence alignment of hMTAP, SsMTAPII and SsMTAP The phosphate (qw) ribose (m) and base (d) binding sites of hMTAP (above the sequence) and of SsMTAP (below the sequence) are indicated Conserved residues between hMTAP and SsMTAPII are highlighted in a grey box SsMTAPII cysteine residues are boxed.
Fig 6 Effect of reducing agents on SsMTAPII thermostability The enzyme (2 lg) was incubated at 80 C for different times in 20 mM Tris ⁄ HCl pH 7.4 in the absence (r) and in the presence of 0.4 M ( ), 0.6 M (m), and 0.8 M (d) dithiothreitol At the time indicated, aliquots were withdrawn and assayed for MTA phosphorylase activ-ity as described under Experimental procedures.
Trang 8inactivate the enzyme suggests that the disulfide(s)
being reduced is quite inaccessible
Effect of mutations on enzyme thermostability
In the absence of the three-dimensional structure of
SsMTAPII, actually under investigation, we utilized
site-directed mutagenesis technique to obtain
informa-tion on the cysteine residues involved in disulfide
bonds We preliminarily selected Cys259, Cys261 and
Cys262 in the C-terminal region of the enzyme and
prepared two mutants, C259S⁄ C261S and C262S We
selected Cys259 and Cys261, as a disulfide
(Cys246-Cys248) has been demonstrated in PfMTAP at the
same conserved positions [16] Moreover, Cys262 was
chosen as a mutagenic target since it is positioned in a
peculiar sequence CXCC It has to be noted that these
three cysteine residues are localized in the C-terminal
region of the enzyme and that this region, as well as
the N-terminal region, are usually highly flexible and
disordered in mesophilic proteins and are thought to
be the first position of the protein which undergoes
denaturation at high temperature [31] Therefore, the
presence of disulfide bonds could increase the stability
of these protein regions
Large-scale production of the two mutant proteins
was performed as described above for the SsMTAPII
Purified mutant proteins showed, under either native
(gel filtration) or denaturing (SDS⁄ PAGE) conditions,
Mrvalues identical to wild-type SsMTAPII and proved
to be fully active demonstrating that the substitutions
were not disruptive
To compare the stabilities of the mutant and
wild-type proteins in terms of thermoactivity and thermal
denaturation we measured their optimal temperatures,
apparent Tm values and residual activities after 1-h
incubation at 90 As reported in Table 1, the
substitu-tion of Cys259, Cys261 and Cys262 with Ser
signifi-cantly affect the thermophilicity and thermostability of
SsMTAPII Both mutated forms showed the same
optimum temperature that is 5C lower than the
wild-type SsMTAPII In contrast, they significantly differ in
their thermostabilities The double mutant, in fact, shows thermal properties lower than those of the single mutant, indicating a significant role of the pair C259-C261 in the stabilization of the protein
We also tested the effect of 0.4 m dithiothreitol on the stability of the double and single mutant at temper-atures where the two proteins exhibited full activity in the absence of the reducing agent In agreement with the results of thermostability experiments, the mutant C259S⁄ C261S is more susceptible than C262S to the reducing agent retaining 55% residual activity after 1-h incubation at 70C in comparison with 63% residual activity displayed by the single mutant after 1-h incuba-tion at 80C (data not shown) These results indicate that, in addition to C259, C261, and C262 other cys-teine residues in SsMTAPII are probably involved in disulfide bonds The results also confirm the important stabilizing role of the pair C259-C261
The reduced thermostability properties of the two mutant proteins with respect to those of the wild-type enzyme and their higher susceptibility to reducing agents indicate that Cys259, Cys261 and Cys262 parti-cipate in the stabilization of the protein, probably forming disulfide bridges The double mutant C259S⁄ C261S deserves particular attention The struc-tural CXC motif of this hypothetical disulfide is un-usual and only a few examples are reported in the literature including CSC in Mengo virus coat protein [32], CDC in Bacillus Ak.1 protease [33], CTC in chap-erone Hsp33 from E coli [34], CGC in yeast thiol oxidase [35], and in MTAP from P furiosus [16] Recently, it has been demonstrated that a CGC motif
in a mutant of E coli thioredoxin reductase [36] dis-plays a disulfide reduction potential that is close to that of protein disulfide isomerase, the most efficient known catalyst of oxidative protein folding These data indicate that the CXC motif is an efficient cata-lyst of disulfide isomerization and that it could play a crucial role in the oxidative protein folding These con-siderations allow us to speculate that the CSC motif in SsMTAPII could also play a similar role in stabilizing the protein disulfide bridges against reductive damage
Comparative kinetic characterization of SsMTAP and SsMTAPII
The occurrence in S solfataricus of SsMTAPII, the second enzyme beside the already isolated and charac-terized SsMTAP devoted to MTA catabolism, promp-ted us to revaluate and define our knowledge about the biochemistry of MTAP in this archaeon There-fore, with the aim of gaining insight into the physio-logical role of SsMTAPII and on its functional
Table 1 Comparative stability features of SsMTAPII and its
mutated forms.
Optimum
temperature (C)
Apparent
Tm(C)
Stability after
1 h at 90 C (% of activity)
Trang 9relationships with SsMTAP, we carried out a
compar-ative kinetic analysis of the two enzymes The Kmand
Vmax values for purine nucleoside substrates in the
presence of saturating concentrations of phosphate
were calculated and typical Michaelis–Menten kinetics
were observed Moreover, the relative efficiency of the
nucleoside substrates was determined by comparing
the respective kcat⁄ Kmratios, which are the best
meas-ure for comparison of the efficiency of product
forma-tion and substrate preference The kinetic parameters
of SsMTAP and SsMTAPII are reported in Table 2
As shown in Table 2, SsMTAP is characterized by
a broad substrate specificity that recognizes both
6-amino- and 6-oxo-purine nucleosides as substrates
The enzyme shows a higher affinity for adenosine (Km
25.4 lm) and inosine (Km 84 lm) compared to
guano-sine (Km113.6 lm) and MTA (Km154.1 lm) Moreover,
SsMTAP displays a lower catalytic efficiency with MTA
(kcat⁄ Km 13.9· 104s)1Æm)1) than in the presence of
other purine nucleosides, suggesting that it could more
appropriately be considered a purine nucleoside
phosphorylase In contrast, inosine and guanosine are
inactive as substrates of SsMTAPII suggesting a
com-pletely different metabolic role for this enzyme Like
SsMTAP, SsMTAPII is able to recognize adenosine
even if the values of affinity and catalytic efficiency for
this substrate are about one order of magnitude lower
than those of SsMTAP indicating that adenosine is not
a physiological substrate of SsMTAPII
An interesting feature of SsMTAPII is the extremely
high affinity for MTA with an apparent Kmof 0.7 lm
This value is about 220-fold lower than that of SsMTAP
and some more, about one order of magnitude lower
than that of hMTAP (Km5 lm) [4], making SsMTAPII
the most MTA-specific enzyme among those reported
in the literature The comparison of the kcat⁄ Kmvalues
for MTA of the two archaeal enzymes clearly indicates
that SsMTAPII is the enzyme physiologically
respon-sible in S solfataricus of the catabolism of MTA
The results of substrate specificity studies are sup-ported by the analysis of the alignment of SsMTAPII sequence with those of SsMTAP and hMTAP repor-ted in Fig 5, where the amino acid residues involved
in the active site of the two enzymes are also indica-ted As expected on the basis of the very high sequence identity between SsMTAPII and hMTAP, the amino acid residues at the active site of the human enzyme are well conserved in SsMTAPII with few substitutions that could be useful to justify why SsMTAPII, unlike hMTAP, is able to recognize adenosine as a substrate
We can first consider that in SsMTAPII the substi-tution of Val233 and Leu237 of the human enzyme with Ala225 and Thr229, respectively, and the absence
of Leu279 could contribute to modify the hydrophobic environment near the 5¢-position of the purine nucleo-side allowing SsMTAPII to bind adenosine
The second observation comes from the evidence that Ile210 of hMTAP is replaced in SsMTAPII by Met204 that in turn is located at the conserved posi-tion of Met181 in SsMTAP sequence It has been dem-onstrated that in SsMTAP this residue provides a key interaction with the ribose moiety Moreover, a methi-onine residue equivalent to Met181 of SsMTAP has been found in all known purine nucleoside phosphory-lase structures [37–40] Therefore, it can be speculated that the substitution of a isoleucine, a residue more suitable for the specific binding of the enzyme with the methylthio group of MTA, with a methionine could allow SsMTAPII, in analogy with SsMTAP, to recog-nize adenosine in addition to MTA
Finally, in SsMTAPII Ser16 and Ser91 replace Thr18 and Thr93, respectively, of human MTAP The substitution of a threonine with a serine residue could make the ribose pocket environment less hydrophobic, thus allowing SsMTAPII to recognize adenosine It is interesting to note that the two threonine residues of human MTAP, which specifically recognizes MTA, are replaced by serine residues in the structurally homo-logous human PNP [40] which instead requires a 5¢-hydroxyl group
On the basis of the reported results, SsMTAPII shows peculiar structural and functional properties The enzyme, in fact, although characterized by the hexameric quaternary structure distinctive of bacterial PNP, exhibits the catalytic properties reminiscent with that of human enzyme In addition, like the homolog-ous MTAP from S solfataricus and P furiosus, SsM-TAPII probably contains disulfide bonds This observation strengthens the current hypothesis that intracellular hyperthermophilic proteins are stabilized
by these covalent links
Table 2 Kinetic parameters of SsMTAP and SsMTAPII Activities
were determined at 70 C as described in Experimental
proce-dures.
K m (lM) k cat (s)1) k cat ⁄ K m (s)1ÆM)1)
SsMTAP
Adenosine 25.4 ± 1 43.6 ± 2 17.2 · 10 5
Guanosine 113.6 ± 6 29.6 ± 2 26.1 · 10 4
SsMTAPII
Adenosine 270.1 ± 13 107 ± 5 39.6 · 10 4
Trang 10Experimental procedures
Bacterial strains, plasmid, enzymes, and
chemicals
Plasmid pET-22b(+) and the NucleoSpin Plasmid kit for
plasmid DNA preparation were obtained from Genenco
(Duren, Germany) E coli strain BL21(kDE3) was
pur-chased from Novagen (Darmstadt, Germany) Specifically
synthesized oligodeoxyribonucleotides were obtained from
MWG-Biotech (Ebersberg, Germany) Restriction
endonuc-leases and DNA-modifying enzymes were obtained from
Takara Bio, Inc (Otsu, Shiga, Japan) Pfu DNA polymerase
was purchased from Stratagene (La Jolla, CA, USA)
[methyl-14C]AdoMet (50–60 mCiÆmmol)1 was supplied by
the Radiochemical Centre (Amersham Bioscience,
Bucking-hamshire, UK) MTA and 5¢-[methyl-14
C]MTA were pre-pared from unlabeled and labeled AdoMet [41] and purified
by HPLC [42] Proteinase K, phenylmethylsulfonyl fluoride,
O-bromoacetyl-N-hydroxysuccinimide and standard
pro-teins used in molecular mass studies were obtained from
Sigma (St Louis, MO, USA) Guanidinium chloride,
dithio-threitol and isopropyl-b-d-thiogalactoside (IPTG) were from
Applichem (Darmstadt, Germany) Sephacryl S-200 and
AH-Sepharose 4B were obtained from Amersham
Pharma-cia Biotech, polyvinylidene fluoride membranes (0.45 mm
pore size) were obtained from Millipore (Bedford, MA,
USA) 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-14
C]MTA [5] Unless otherwise sta-ted, the standard incubation mixture contained the
following: 20 lmol potassium phosphate buffer, pH 7.4,
80 nmol of [methyl-14C]MTA (6.5· 105
c.p.m.Ælmol)1), and the enzyme protein in a final volume of 200 lL The
incubation was performed in sealed glass vials for 5 min at
70C, 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 H20 5-[methyl-14
C]Methylthio-ribose-1-phosphate produced was eluted with 2.5 mL of
0.01 m HCl 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 70C, the reaction mixture was preincubated
for 2 min without the enzyme that was added immediately
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 (nucleoside + purine base) absorbance integrated peak areas An
Ultrasil-CX column (Beckman) eluted with 0.05 m ammonium for-mate, pH 3 at a flow rate of 1 mLÆmin)1 was used when adenosine and⁄ or guanosine were the substrates of the reac-tion In these experimental conditions the retention times of adenosine and adenine, guanosine and guanine were 7.3 min and 12.4 min, and 4.2 min and 6 min, respectively When the assays were carried out in the presence of inosine as sub-strate, 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 in H2O The retention times of inosine and hypoxantine were 10.5 min 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% trifluoroacetic acid in H2O The retention times of MTA and adenine were 10 min and 4.2 min, respectively
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
Determination of kinetic constants Homogeneous preparations of SsMTAPII and SsMTAP [5] were used for kinetic studies The purified enzymes gave a linear rate of reaction for at least 10 min at 70C, thus, an incubation time of 5 min was than employed for kinetic experiments All enzyme reactions were performed in tripli-cate Kinetic parameters were determined for both enzymes
by varying the concentrations of purine nucleosides in the assay mixture in the presence of 100 mm phosphate Kinetic parameters were determined from Lineweaver–Burk plots of initial velocity data Kmand Vmaxvalues were obtained from linear regression analysis of data fitted to the Michaelis– Menten equation Values given are the average from at least three experiments with standard errors The kcatvalues were calculated by dividing Vmaxby the total enzyme concentra-tion Calculations of kcatwere based on an enzyme molecular mass of 160 kDa for SsMTAP and 180 kDa for SsMTAPII
Analytical methods for protein Proteins were assayed by the Bradford method [43] using bovine serum albumin as standard The molecular mass of the native protein was determined by gel filtration at 20C
on a calibrated Sephacryl S-200 column (2.2· 95 cm) equili-brated with 10 mm Tris⁄ HCl, pH 7.4, containing 0.3 m