Purine nucleoside phosphorylases from hyperthermophilic
Archaea require a CXC motif for stability and folding
Giovanna Cacciapuoti, Iolanda Peluso, Francesca Fuccio and Marina Porcelli
Department of Biochemistry and Biophysics ‘F. Cedrangolo’, Second University of Naples, Italy
Introduction
In the fascinating field of protein biochemistry, ther-
mostability and folding comprise important factors
that are currently gaining wide attention. Disulfide
bonds represent an important structural feature of
many proteins, especially extracellular ones. They not
only stabilize protein structures by lowering the
entropy of the unfolded polypeptide, but also are
required for the proper folding and biological activity
of several proteins. Disulfide bond formation occurs in
the endoplasmic reticulum and mitochondrial inter-
membrane space of eukaryotes and in the periplasm of
prokaryotes [1]. Disulfide bonds are a typical feature
of secretory proteins and are considered to contribute
significantly to their overall stability [2]. By contrast,
in intracellular proteins from well-known organisms,
and as a result of the reductive chemical environment
inside the cells [3], the presence of these covalent links
is limited to proteins involved in the mechanism of
the response to redox stress [4] or to proteins catalyz-
ing oxidation–reduction processes [1,5]. Despite this
classical view, recent computational, structural and
biochemical studies have highlighted the critical role of
Keywords
5¢-deoxy-5¢-methylthioadenosine
phosphorylase; CXC motif and oxidative
protein folding; disulfide bonds;

containing peptides are also able to reactivate scrambled RNaseA. The
data obtained in the present study represent the first example of how the
CXC motif improves both stability and folding in hyperthermophilic
proteins with disulfide bonds.
Abbreviations
AdoMet, S-adenosylmethionine; GdnCl, guanidinium chloride; GSH, glutathione; GSSG, glutathione disulfide; MTA, 5¢-deoxy-5¢-
methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PDI, protein disulfide isomerase; PfCGC, NH
2
-RRCGCKD-
COOH; PfPNP, purine nucleoside phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; sRNaseA, scrambled
RNaseA; SsCSC, NH
2
-GSCSCCN-COOH; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus.
FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5799
these covalent links in the structural stabilization of
intracellular proteins in some hyperthermophilic
Archaea and Bacteria [6–12].
The abundance of disulfides observed across hyper-
thermophilic organisms also has stimulated research
into the identification of the biochemical mechanisms
related to disulfide maintenance. It was recently dem-
onstrated that specific protein disulfide oxidoreducta-
ses, which are structurally and functionally related to
protein disulfide isomerase (PDI) [13], play a key role
in intracellular disulfide shuffling in hyperthermophilic
proteins [14].
Two enzymes, 5¢-deoxy-5¢-methylthioadenosine pho-
sphorylase II from Sulfolobus solfataricus (SsMTAPII)
[10,11] and purine nucleoside phosphorylase from
Pyrococccus furiosus (PfPNP) [12], have been isolated

affects both their thermodynamic and kinetic stability
[10,12], suggesting the involvement of the cysteine pair
in the thermal stabilization of both enzymes.
In the present study, we demonstrate that the CXC
motif of SsMTAPII and PfPNP plays an important
functional role in the oxidative folding of these
enzymes and that two short CXC-containing peptides
with amino acid sequences corresponding to those
present in the C-terminal region of SsMTAPII and
PfPNP, respectively, act in vitro as functional mimics
of PDI. The presence of a CXC motif in hyperthermo-
philic proteins with multiple disulfide bonds, such as
SsMTAPII and PfPNP, represents the first example of
a new molecular strategy adopted by these enzymes to
improve their stability and to preserve their folded
state under extreme conditions.
Results and Discussion
SsMTAPII and PfPNP require a CXC motif to
preserve their folded state
SsMTAPII and PfPNP are highly thermoactive, with
an optimum temperature of 120 °C, and extremely
thermostable, with apparent T
m
values of 112 °C and
110 °C, respectively. These enzymes are also character-
ized by a remarkable kinetic stability and resistance to
many chemicals, including SDS and guanidinium chlo-
ride (GdnCl) [10,12]. As demonstrated by structural
and biochemical studies, SsMTAPII and PfPNP utilize
multiple intrasubunit disulfide bridges as a principal

5800 FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS
logous to SsMTAPII and PfPNP. It is interesting to
note that a CGC motif at the C-terminus of the yeast
thiol oxidase Erv2p was found to be involved in the
exchange of the de novo synthesized disulfide bridge
with substrate protein [25]. Moreover, it was demon-
strated that a synthesized CGC peptide and a CGC
motif in a mutant of E. coli thioredoxin were func-
tional mimics of PDI [26]. Taken together, these data
allowed us to speculate that the CXC motif in SsM-
TAPII and PfPNP could act as a redox reagent and
exert its stabilizing role by rescuing, in analogy with
PDI, any possible damage of the other disulfide bonds
of the protein. To demonstrate the active role of CXC
motif in the oxidative folding process, we carried out
the unfolding of SsMTAPII, PfPNP and their CXC-
lacking mutants by incubation for 22 h at 25 °C with
6 m GdnCl in 20 mm Tris ⁄ HCl (pH 7.4), containing
30 mm dithiothreitol. The reversibility of the GdnCl-
induced unfolding was then examined by assaying
the catalytic activity after complete removal of the
denaturant.
As shown in Fig. 1, SsMTAPII and PfPNP are able
to refold with a recovery of catalytic activity of 59%
and 90%, respectively, compared to their control
enzymes. By contrast, lower values of reactivation were
observed for SsMTAPIIC259S ⁄ C261S and PfPNPC
254S ⁄ C256S, the two CXC-lacking mutants (25% and
46% activity, respectively). These results demonstrate
that the CXC motif is necessary to obtain almost com-

2
-GSCSCCN-COOH (SsCSC) and
NH
2
-RRCGCKD-COOH (PfCGC) act as in vitro
catalysts of oxidative protein folding
PDI, the most efficient known catalyst of oxidative
folding, is a multifunctional eukaryotic enzyme that
utilizes the active site motif CGHC to catalyze the for-
mation of native disulfides and the rearrangement of
incorrect disulfide bonds, especially those within kineti-
cally trapped, structured folding intermediates [13]. In
recent years, interest in protein folding in vitro has
expanded rapidly, mainly focusing on the production,
in bacteria, of disulfide-containing proteins with poten-
tial biotechnological applications. Therefore, increased
attention has been paid to the design and synthesis of
novel, small-molecule reagents that could improve the
efficiency of the oxidative folding process. Recently, on
the basis of the physical properties of PDI, a variety
of CXXC peptides have been synthesized and assayed
[27]. The active site of PDI has also been modeled as a
SsMTAPII
SsMTAPII C259S/C261S
PfPNP
PfPNP C254S/C256S
100
60
80
SsMTAPII

incorrect protein disulfide followed by a thiol–disulfide
interchange within the substrate, leading in turn to a
native disulfide. Two CXC-containing peptides, namely
SsCSC and PfCGC, whose amino acid sequences are
identical to those present in the C-terminal region of
SsMTAPII and PfPNP, respectively, have been synthe-
sized and their disulfide isomerase activity has been
assayed utilizing the partially refolded forms of
SsMTAPII, PfPNP and their CXC-lacking mutants as
substrates. As shown in Fig. 3, both peptides are
involved in the oxidative folding of these enzymes with
a concomitant recovery of their catalytic activity.
Indeed, after 22 h of incubation in the presence of
SsCSC and PfCGC, the enzymatic activity of
SsMTAPII and its mutant, expressed as a percentage
of their control enzymes, reaches 86.8% and 51.8%,
and 68% and 49.3%, respectively (Fig. 3A). Similar
results were obtained when the unfolded ⁄ refolded
forms of PfPNP and its mutant were assayed under
the same experimental conditions (Fig. 3B). It is inter-
esting to note that, although PfPNP and its mutant
show a higher reactivation values than SsMTAPII and
its mutant (Fig. 3), the ratio of these values with
respect of their control enzymes is similar, thus indicat-
ing that the efficiency of the process is comparable.
These data demonstrate that the CXC motif of SsM-
TAPII and PfPNP is active, even when isolated from
the proteins, and that it is able to induce the in vitro
oxidative folding of these enzymes.
To further confirm the ability of SsCSC and PfCGC

C
U/R + PfCGC
Relative activity (%)
Fig. 3. Effect of CXC-containing peptides on
the reactivation of refolded SsMTAPII,
PfPNP and their mutants. SsMTAPII, PfPNP
and their mutants, partially refolded after
GdnCl-induced unfolding, (U ⁄ R), were incu-
bated for 22 h at 30 °C in the presence of
various oxidative folding catalysts (reactiva-
tion assay). The catalytic activity of (A) SsM-
TAPII and SsMTAPIIC259S ⁄ C261S and (B)
PfPNP and PfPNPC254S ⁄ C256S was then
measured under standard assay conditions.
The activity of control enzymes was
expressed as 100%. Each value is the
average of three separate experiments.
CXC and oxidative protein folding G. Cacciapuoti et al.
5802 FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS
as PDI, catalyzing the rearrangement of incorrect
disulfide bonds in a protein substrate. It is interesting
to note that SsCSC displays a higher oxidative folding
activity than PfCGC (Figs 3 and 4), suggesting that
the presence in SsCSC of a third thiol in the sequence
CXCC could most likely enhance the reactivity toward
disulfide bonds.
In conclusion, the results obtained in the present
study provide insight into the variety of molecular
mechanisms utilized for stabilizing folded proteins under
extreme thermal environments and allow us to speculate

Assays of enzyme activity
PNP activity was determined by monitoring the formation
of purine base from the corresponding nucleoside by HPLC
using a Beckman System Gold apparatus (Beckman Coul-
ter, Fullerton, CA, USA). The assay was carried out as
described previously [12].
MTAP activity was determined by monitoring the forma-
tion of [methyl-
14
C]5-methylthioribose-1-phosphate from
5¢-[methyl-
14
C]MTA [10]. In all enzymatic assays, 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.
GdnCl-induced unfolding and refolding
SsMTAPII, PfPNP and their respective CXC-lacking
mutants (final concentration 0.4 mgÆmL
)1
) were incubated
for 22 h at 25 °C in the presence of 6 m GdnCl in 20 mm
Tris ⁄ HCl (pH 7.4) containing 30 mm dithiothreitol. Unfold-
ing was probed by recording the intrinsic fluorescence emis-
sion. To test the reversibility of the process, the refolding
was started by a 20-fold dilution of the unfolding mixture
in Tris ⁄ HCl 20 mm (pH 7.4). The refolded enzyme, after
extensive dialysis against Tris ⁄ HCl 20 mm (pH 7.4) until
complete removal of GdnCl, was analyzed by catalytic

, PDI; d, SsCSC; , PfCGC.
Each value is the average of three separate experiments.
G. Cacciapuoti et al. CXC and oxidative protein folding
FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5803
the enzymes catalyzed by PDI (final concentration 0.11 lm)
under the same experimental conditions.
Reactivation of sRNaseA
Disulfide isomerase activity was assayed as described previ-
ously [30] by monitoring the reactivation of sRNaseA, a
fully oxidized protein containing a random distribution of
its four disulfide bonds. SsCSC and PfCGC were first
reduced with a five-fold excess of dithiothreitol in 50 mm
Tris ⁄ HCl (pH 7.4) for 10 min at 30 °C and then incubated
for 1 h at 30 °C in a reactivation mixture containing 0.1 m
Tris-acetate buffer (pH 8.0), 2 mm EDTA, a glutathione
redox buffer (1 mm GSH, 0.2 mm GSSG), 2 mm CXC-pep-
tide, and sRNaseA (0.5 mgÆmL
)1
in 10 mm acetic acid; final
concentration 19.6 lm). cCMP at a final concentration of
4mm was then added and A
296
, as a result of RNase-cata-
lyzed cCMP hydrolysis, was monitored continuously for
210 min at 30 °C. The positive control was the reactivation
of sRNaseA catalyzed by PDI (final concentration 4 lm).
The control for the non-enzymatic reactivation of sRNaseA
was represented by the same mixture without the addition
of any oxidative folding catalyst.
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