Tài liệu Báo cáo khoa học: Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus – biochemical characterization and homology modeling doc

Pyrimidine-specific ribonucleoside hydrolase from the
archaeon Sulfolobus solfataricus – biochemical
characterization and homology modeling
Marina Porcelli
1,2
, Luigi Concilio
1
, Iolanda Peluso
1
, Anna Marabotti
3
, Angelo Facchiano
3
and
Giovanna Cacciapuoti
1
1 Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita
`
di Napoli, Italy
2 Consorzio Interuniversitario Biostrutture e Biosistemi ‘INBB’, Rome, Italy
3 Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy
Nucleoside hydrolases (NHs; EC 3.2.2.–) catalyze the
irreversible hydrolysis of the N-glycosidic bond of
b-ribonucleosides, forming ribose and the free purine
or pyrimidine base [1–3]. All characterized members
are metalloproteins with a unique central b-sheet
topology and a cluster of aspartate residues
(DXDXXXDD motif) at the N-terminus of the
enzyme [2–5].
In nature, a widespread distribution of NHs in dif-
ferent protozoa [6–11], bacteria [12–14], yeasts [15–17],

insects [18] and mesozoa [19] is observable. Genes con-
taining the characteristic NH structural motif have
been also found in plants [20,21], amphibians and
fishes [3].
Nucleoside hydrolases play a well-established
key role in the purine salvage pathway of parasitic
Keywords
homology modeling; hyperthermostability;
nucleoside hydrolase; nucleoside
metabolism; Sulfolobus solfataricus
Correspondence
M. Porcelli, Dipartimento di Biochimica e
Biofisica ‘F. Cedrangolo’, Seconda Universita
`
di Napoli, Via Costantinopoli 16,
Napoli 80138, Italy
Fax: +39 081 5667519
Tel: +39 081 5667545
E-mail:
(Received 23 November 2007, revised 11
February 2008, accepted 20 February 2008)
doi:10.1111/j.1742-4658.2008.06348.x
We report the characterization of the pyrimidine-specific ribonucleoside
hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus
(SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and
expressed in Escherichia coli and the recombinant protein was purified to
homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes
uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity
with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The align-
ment of the amino acid sequences of SsCU-NH with nucleoside hydrolases

whose 3D structures have been solved indicates that the amino acid resi-
dues involved in the calcium- and ribose-binding sites are preserved.
SsCU-NH is highly thermophilic with an optimum temperature of 100 °C
and is characterized by extreme thermodynamic stability (T
m
= 106 °C)
and kinetic stability (100% residual activity after 1 h incubation at 90 °C).
Limited proteolysis indicated that the only proteolytic cleavage site is local-
ized in the C-terminal region and that the C-terminal peptide is necessary
for the integrity of the active site. The structure of the enzyme determined
by homology modeling provides insight into the proteolytic analyses as well
as into mechanisms of thermal stability. This is the first nucleoside hydro-
lase from Archaea.
Abbreviations
Cf, Crithidia fasciculata; CU-NH, pyrimidine-specific ribonucleoside hydrolases; Ec, Escherichia coli; IAG-NH, purine-specific inosine-
adenosine-guanosine nucleoside hydrolases; IG-NH, 6-oxo-purine-specific inosine-guanosine nucleoside hydrolases; IPTG, isopropyl thio-b-
D-galactoside; IU-NH, purine-nonspecific inosine-uridine nucleoside hydrolases; Lm, Leishmania major; MTA, 5¢-methylthioadenosine; MTAP,
5¢-methylthioadenosine phosphorylase; MTAPII, 5¢-methylthioadenosine phosphorylase II; MTI, methylthioinosine; NH, nucleoside hydrolase;
NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PVDF, poly(vinylidene fluoride); Ss, Sulfolobus solfataricus;
Tv, Trypanosoma vivax.
1900 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
protozoa [6–11]. In these organisms, the nucleoside sal-
vage pathway is vital because, in contrast to most
other living organisms, they lack a de novo biosynthetic
pathway for purines [6–11,22]. All protozoa therefore
utilize salvage enzymes such as NHs and phospho-
ribosyltransferases to form nucleotides [22,23]. Because
neither NH activity, nor the encoding genes have ever
been detected in mammals, the parasitic NHs have
been studied extensively in recent years as attractive

potential targets for drug development [24,25]. Indeed,
highly potent NH inhibitors could be very effective
against protozoan infection.
According to their substrate specificity NHs can be
classified into different subclasses: the purine-non-
specific inosine-uridine nucleoside hydrolases (IU-NH)
[3,7,9,26], the purine-specific inosine-adenosine-guano-
sine nucleoside hydrolases (IAG-NH) [3,11,27,28],
the pyrimidine-specific nucleoside hydrolases (CU-NH)
[3,13,15–17,29,30] and the 6-oxo-purine-specific ino-
sine-guanosine nucleoside hydrolases (IG-NH) [3,31].
Recently, a number of NHs have been fully character-
ized and the crystal structures were also solved
[9,11,19,26,29,30].
Ribonucleosides are predominantly metabolized by
nucleoside phosphorylases (NP), which catalyze the
reversible phosphorolytic cleavage of the glycosidic
bond yielding ribose 1-phosphate and the correspond-
ing free base [32–34]. In our laboratory, two NPs have
been purified and extensively characterized from
Sulfolobus solfataricus [35–38], an extreme thermo-
acidophilic microorganism optimally growing at 87 °C,
belonging to Archaea, the third primary domain [39].
Hyperthermophilic Archaea are of extreme interest for
understanding the molecular mechanisms of structural
and functional adaptation of proteins to extreme tem-
peratures and also for the peculiar substrate specificity
of their enzymes that provide unique models for study-
ing enzyme evolution in terms of structure, specificity
and catalytic properties [40–42].

To elucidate the mechanisms by which hyperthermo-
philic enzymes acquire their unusual thermostability
and to increase our knowledge on the structure of
NHs, we carried out the expression, purification and
physicochemical characterization of a NH from S. sol-
fataricus, (SsCU-NH), aiming to elucidate the struc-
ture ⁄ function ⁄ stability relationships in this enzyme and
to explore its biotechnological applications. A detailed
kinetic investigation was also performed to define
the substrate specificity of SsCU-NH and to study
the functional role played by this enzyme in
the purine ⁄ pyrimidine nucleoside metabolism. Finally,
the 3D structure of the enzyme was constructed by
homology modeling using the crystal structure of
Escherichia coli pyrimidine-specific NHs Yeik [29] and
Ybek [30] as templates. The structure provided insight
into the active site architecture of SsCU-NH as well as
into the features of the protein that may contribute to
its thermostability. This is the first example of a NH
reported in Archaea.
Results and Discussion
Analysis of SsCU-NH gene and primary structure
comparison
The analysis of the complete sequenced genome of
S. solfataricus revealed an ORF (SSO0505) encoding a
311 amino acid protein homologous to a NH, which is
annotated as iunH-1. The putative molecular mass of
the protein predicted from the gene was 35.21 kDa
and the estimated isoelectric point was 5.17.
The coding region starts with an ATG triplet at the

position 438552 of the S. solfataricus genome. The first
stop codon TAG is encountered at the position 439485
and is preceded by a TTT, which codes for phenyl-
alanine. Upstream from the coding region and 14 bp
before the starting codon, there is a stretch of purine-
rich nucleosides (GTGGTAGA) that may function as
the putative ribosome-binding site [43]. Putative pro-
moter elements box A and box B, which are in good
agreement with the archaeal consensus [43], are found
close to the putative transcription start site. A hexa-
nucleotide with the sequence TTTAAG similar to box
A is located 30 bp upstream from the start codon and
resembles the TATA box, which is involved in binding
the archaeal RNA polymerase [43]. A putative box B
(TTGT) is 16 bp upstream from the start codon.
Finally, a pyrimidine-rich region (TTTGAATTTTTA),
strictly resembling the archaeal terminator signal [43],
is localized 11 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 from S. solfataricus
or from other Archaea.
Comparison of the deduced primary structure of
SsCU-NH with enzymes present in GenBank database
reveals the highest similarity with the hypothetical NH
from Sulfolobus tokodaii (64% sequence identity), from
Sulfolobus acidocaldarius (60% sequence identity), and
with a second hypothetical NH from S. solfataricus
(43% sequence identity). Among the related enzymes
isolated from various sources, SsCU-NH shows signifi-

cant sequence identity with pyrimidine-specific NHs
from E. coli YeiK (34%) and YbeK (30%).
Figure 1 shows the multiple sequence alignment
of SsCU-NH with homologous enzymes whose 3D
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1901
structures have been solved, such as the purine-non-
specific NHs from Crithidia fasciculata (CfIU-NH)
[26] and Leishmania major (LmIU-NH) [9], the
pyrimidine-specific NHs from E. coli such as YeiK
(Ec-YeiK) [29] and YbeK (Ec-YbeK) [30], and with
the purine-specific NH from Trypanosoma vivax
(TvIAG-NH) [11].
The analysis of the sequence alignment shows that
the amino acid residues involved in the calcium-bind-
ing site and in the ribose binding site of these enzymes
are well conserved in SsCU-NH. Figure 1 also com-
pares the nucleoside base specificity in the active sites
of the TvIAG-NH and Ec-YeiK. In this regard, it
should be noted that TvIAG-NH binds the purine ring
Fig. 1. Multiple sequence alignment of SsCU-NH, CfIU-NH, LmIU-NH, Ec-YeiK, Ec-YbeK and TvIAG-NH. The calcium ( ) ribose (d) and base
(+) binding sites of Ec-YeiK are indicated above the alignment. The residues at the active site of TvIAG-NH are indicated below the sequence
with the same symbols. Identical and conserved residues are highlighted in dark and pale gray respectively. DXDXXXDD motif is shown in
white lettering on a black background. Numbers on the right are the coordinates of each protein.
CU-NH from S. solfataricus M. Porcelli et al.
1902 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
with N12, D40, W83 and W260, whereas the base-
binding pocket of Ec-YeiK is composed of N80, I81,
H82, F159, F165, T223, Q227, Y231 and H239. From
the comparison, it appears that SsCU-NH maintains

the same overall active site organization of Ec-YeiK as
for the base binding site.
Enzyme expression, purification and properties
To overproduce SsCU-NH, the gene was amplified by
PCR and cloned into pET-22b(+) under the T
7
RNA
polymerase promoter. The gene sequence was found to
be identical with the published one [43a].
Recombinant SsCU-NH was expressed in a soluble
form in E. coli BL21 cells harboring pET-SsCU-NH.
A good level of expression was obtained by optimiz-
ing both the growth time of the transformed cells and
the induction time with isopropyl thio-b-d-galactoside
(IPTG). The most favorable conditions for the
expression of the enzyme were found to be when
IPTG was added at A
600
= 3.0 and when the induc-
tion was prolonged for 16 h. Therefore, these condi-
tions were chosen for large-scale production and
approximately 10 g of wet cell paste was obtained
from 1 L of culture.
SDS ⁄ PAGE analysis of cell-free extract of induced
cells revealed an additional band of approximately
35 kDa, which corresponded 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 SsCU-NH production
in E. coli BL21 cells harboring pET-SsCU-NH, was

found to be of 170 nmol of uridine cleavedÆmin
)1
Æmg
)1
at 80 °C, confirming that the SsCU-NH gene had been
cloned and expressed.
Direct evidence that this putative NH is present
in S. solfataricus comes from experimental results
obtained measuring the nucleoside hydrolase activity
of the crude extract after extensive dialysis against
10 mm Tris ⁄ HCl (pH 7.4) to make the cell homogenate
phosphate-free and to assure that the degradation of
nucleoside substrate cannot be ascribed to NP activity.
The results obtained indicate that NH activity of
S. solfataricus cells is approximately 10 nmol of uri-
dine cleavedÆmin
)1
Æmg
)1
at 80 °C.
Recombinant SsCU-NH was easily purified to
homogeneity by a fast and efficient two-step procedure
that utilizes a heat treatment and affinity chromatogra-
phy on 5¢-methylthioinosine (MTI)-sepharose. Approx-
imately 2 mg of the recombinant enzyme with a 20%
yield was obtained from 1 L of culture (data not
shown). No processing occurred at the amino terminus
of the enzyme in the E. coli system, as demonstrated
by sequence determination of the first ten amino acids
of SsCU-NH.

SDS ⁄ PAGE of the enzyme reveals a single band
with an apparent molecular mass of 33 ± 1 kDa,
which is in fair agreement with the expected mass cal-
culated from the amino acid sequence. The identity of
the protein was checked by N-terminal sequencing and
was confirmed by MALDI-MS analysis of the HPLC
purified protein.
The molecular mass of SsCU-NH was estimated to
be 140 ± 7 kDa by size exclusion chromatography,
which indicated a homotetrameric structure in solu-
tion. Therefore, on the basis of its quaternary struc-
ture, SsCU-NH is a member of the tetrameric group
of NHs together with the structurally characterized
NHs from parasitic protozoa, including NHs from
Crithidia fasciculata [7,26], Leishmania major [9] and
Leishmania donovani [10], from Bacteria, such as NHs
from E. coli YeiK and YbeK [29,30], and from the hel-
minth parasite Caenorhabditis elegans [19].
Like all other characterized NHs, and in agreement
with the results of the comparative primary structure
analysis, SsCU-NH is a Ca
2+
-dependent enzyme. After
1 h of incubation with EDTA (5 mm), the enzyme
activity was reduced to <0.05% and was restored by
the addition of 20 mm CaCl
2
(data not shown), indi-
cating that Ca
2+

is required in maintaining the active
site structure.
Substrate specificity and kinetic characterization
With the aim of gaining insight on the physiological
role of SsCU-NH, we carried out a detailed kinetic
characterization of this enzyme. The enzymatic charac-
terization defines SsCU-NH as a pyrimidine-specific
NH. This enzyme was completely inactive towards
adenosine and guanosine. SsCU-NH, in analogy with
Ec-Yeik enzyme, is specific for uridine and cytidine
and is unable to hydrolyze the deoxyribonucleosides
such as thymidine and deoxycytidine. This evidence
confirms a common characteristic for all NHs that
bind the 2¢-hydroxyl of the ribose ring with specific
hydrogen bonds by the conserved Asp residues in the
active site. In addition, SsCU-NH is not active with
nucleoside 5¢-phosphates as substrate and the catalytic
efficiency towards inosine is at least 100-fold below
that for uridine.
Initial velocity studies carried out with increasing
concentrations of pyrimidine nucleosides gave typical
Michaelis–Menten kinetics. The recombinant enzyme
shows Michaelis constants for uridine and cytidine of
the same order of magnitude, within the experimental
errors, with K
m
values of 310 lm and 970 lm respec-
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1903
tively. Moreover, as shown in Table 1, the relative effi-

ciency of these two substrates, determined by comparing
the respective k
cat
⁄ K
m
ratios, was also comparable.
The results of substrate specificity studies are sup-
ported by the analysis of the sequence alignment
reported in Fig. 1. As expected on the basis of the rel-
atively high sequence identity (34%), the hypothetical
active site of SsCU-NH is very similar to Ec-YeiK and
only few key residue changes are observable. The
occurrence in S. solfataricus of SsCU-NH, prompted
us to revaluate and define our knowledge about
the biochemistry of nucleoside metabolism in this
archaeon.
Depending on the organism, the release of the bases
from nucleosides can occur through actions of NP
and ⁄ or NH. Two different NPs have been isolated and
characterized from S. solfataricus,5¢-methylthioadeno-
sine phosphorylase (SsMTAP, gene number SSO2706)
[35,36] and 5¢-methylthioadenosine phosphorylase II
(SsMTAPII, gene number SSO2343) [37,38]. On the
basis of their structural and functional features,
SsMTAP and SsMTAPII are two completely different
enzymes. SsMTAP is a hexameric protein with high
sequence identity to E. coli purine nucleoside phos-
phorylase (PNP) and with a broad substrate specificity
recognizing either 6-oxo or 6-amino purine nucleosides
as substrates. On the other hand, SsMTAPII, although

characterized by the hexameric quaternary structure
distinctive of bacterial PNP, exhibits catalytic proper-
ties reminiscent with human MTAP, recognizing only
6-aminopurine nucleoside as substrates and showing
an extremely high affinity for 5¢-methylthioadenosine
(MTA).
Homology-based database searches in the complete
genomic sequence of S. solfataricus revealed the pres-
ence of an additional putative NP gene (SSO1519).
To accomplish detailed structural and functional
studies on this enzyme and to verify its substrate
specificity, we carried out the expression of the pro-
tein in E. coli. The catalytic activity of recombinant
enzyme was assayed utilizing purine and pyrimidine
ribonucleosides or deoxyribonucleosides as substrate
of the phosphorolytic reaction. By contrast to our
expectations, no NP activity was detectable with all
nucleosides tested, even when modifying the assay
conditions in different ways. Therefore, we think that
the annotation of this gene as putative NP is not
correct. On the basis of the obtained results, SsCU-
NH is the only known enzyme physiologically
involved in the pyrimidine nucleoside catabolism in
this archaeon.
Thermal properties and limited proteolysis
The temperature dependence of the activity of SsCU-
NH in the range 40–130 °C is shown in Fig. 2. The
enzyme is highly thermoactive; its activity increased
sharply up to the optimal temperature of 100 °C and a
50% activity was still observed at 110 °C. This behav-

ior led to a discontinuity in the Arrhenius plot at
approximately 80 °C, with two different activation
energies, suggesting that conformational changes can
occur in the protein structure around this temperature.
To study the thermodynamic stability of SsCU-NH,
we measured the residual activity after 10 min of incu-
bation at increasing temperature. The corresponding
diagram reported in Fig. 3A is characterized by a
sharp transition that allowed us to calculate an appar-
ent melting temperature of 106 °C. The resistance of
SsCU-NH to irreversible heat inactivation processes
was monitored by subjecting the enzyme to prolonged
incubations in the temperature range 90–110 °C and
by measuring the residual activity under standard con-
ditions. As observed in Fig. 3B, the enzyme decay
obeys first-order kinetics. The results obtained indicate
that SsCU-NH is characterized by a notably high
kinetic stability retaining full activity after 1 h of incu-
bation at 90 °C and showing half-lives of 37, 24 and
5 min at 100, 105 and 110 °C respectively.
Table 1. Kinetic parameters of SsCu-NH. Activities were deter-
mined at 80 °C as described in the Experimental procedures.
K
m app
(lM) k
cat
(s
)1
) k
cat

⁄ K
m app
(s
)1
ÆM
)1
)
Uridine 310 ± 20 7.1 ± 0.2 (22.9 ± 0.8) · 10
3
Cytidine 970 ± 50 39.4 ± 1.2 (40.6 ± 0.8) · 10
3
Fig. 2. The effect of temperature on SsCU-NH activity. The activity
observed at 100 °C is expressed as 100%. The assay was per-
formed as indicated in the Experimental procedures. Arrhenius plot
is reported in the inset; T, temperature (°K).
CU-NH from S. solfataricus M. Porcelli et al.
1904 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
To explore the correlation between the resistance to
proteolysis and the conformational protein stability
and to obtain information about the flexible regions of
SsCU-NH exposed to the solvent and susceptible to
proteolytic attack, we subjected the enzyme to limited
proteolysis. The application of limited proteolysis can
often provide useful information about conformational
changes resulting in protection of the cleavage sites or
uncovering new sites [44–46]. SsCU-NH was com-
pletely resistant to trypsin, whereas proteinase K, sub-
tilisin and thermolysin were able to cleave the enzyme.
Therefore, proteolytic degradation of SsCU-NH was
investigated by measuring the residual activity after

incubation with proteinase K or subtilisin at 37 °Cor
with thermolysin at 60 °C followed by SDS ⁄ PAGE of
the digested material. All these proteases produced
essentially the same results, and only the results for
proteinase K are discussed. A protein band with an
apparent molecular mass of approximately 10.6 kDa
less than that of SsCU-NH appears as the proteolysis
proceeds and a concomitant decrease of catalytic activ-
ity was observed (data not shown). The analysis of the
proteolytic fragment by Edman degradation showed
that the amino terminus was preserved, thus indicating
that the proteolytic cleavage site is localized in the
C-terminal region and that the C-terminal peptide of
SsCU-NH is necessary for the integrity of the active
site. These results confirm the conclusions drawn from
the analysis of the sequence alignment reported in
Fig. 1, which highlights the presence of one hypotheti-
cal pyrimidine base-binding site in the C-terminal
region of the enzyme, as well as one Ca
2+
-binding site.
Nevertheless, no substrate-protection against proteoly-
sis was observed.
Structural overview of SsCU-NH
The structures of two proteins from E. coli (YeiK and
YbeK) belonging to the subclass of pyrimidine-specific
NHs were recently obtained by X-ray crystallography
(PDB files 1Q8F and 1YOE, respectively) [29,30].
YbeK and YeiK were retrieved from the BLAST anal-
ysis as suitable templates to model the structure of

SsCU-NH. The optimal alignment between SsCU-NH
and its structural templates was obtained by extracting
the sequences of the target and the templates from a
global alignment with 30 sequences belonging to the
NH family. The type and position of the predicted sec-
ondary structures, with few exceptions, are superim-
posed on those present in the templates, supporting
the correctness of the final alignment that was used to
create the structure of the monomeric SsCU-NH (data
not shown).
Among the ten models obtained using the two ver-
sions of the program modeller, we chose the best one
both in terms of stereochemical parameters (91.1%
of the amino acids in the most favored regions of
the Ramachandran plot) and ProsaII z-score
(z-score = ) 10.30, analogous to that of the template,
which is equal to )10.84). Experimental evidence con-
firms that SsCU-NH is a tetramer. Therefore, we
assembled its oligomeric form using the 3D structure
of YeiK enzyme as template.
The superposition of the tetrameric model with its
template YeiK shows an RMSD of 0.53 A
˚
, indicating
that no major differences are present between target
and template in terms of global architecture (Fig. 4A).
Each subunit of SsCU-NH is made of a central b-sheet
composed of seven parallel and one antiparallel
Fig. 3. Thermostability of SsCU-NH. (A) Residual SsCU-NH activity after 10 min of incubation at the temperatures shown. Apparent T
m

is
shown in the inset. (B) Kinetics of thermal inactivation of SsCU-NH as a function of incubation time. The enzyme was incubated at 90 °C
(
¤), 100 °C( ), 105 °C( ), 107 °C(·) and 110 °C(d) for the time indicated. Aliquots were then withdrawn and assayed for the activity as
described in the Experimental procedures.
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1905
b-strands, flanked by a-helices (Fig. 4B). Loops G63-
V75 and G80-A101, which are connected by a short
a-helix structure, are thought to be segments with high
conformational flexibility because they undergo very
large conformational changes in YeiK as the substrates
bind to the enzyme, and determine the transition from
the ‘open’ to the ‘closed’ state, with obvious implica-
tions on enzyme function and catalysis [29]. Loop 63–
75 is at the interface between the monomers A ⁄ B and
C ⁄ D, whereas segment 80–101 is pointing towards the
exterior of the protein. Other unstructured segments
are G148-E162, V228-D238 and D275-N288. The first
one points towards the interior of the tetramer. The
other two are located near the interface between
monomers A ⁄ C and B ⁄ D.
The structure of SsCU-NH was analyzed in terms of
the results obtained from limited proteolysis of the
protein. Based on the proteolysis data, the cleavage
point should lie in loop 228–238 between strand S8
and helix H11, which is exposed to solvent (Fig. 4B).
Moreover, the first part of this segment (228–232) pro-
trudes towards the exterior of the tetramer near loop
275–288 of the opposite monomer. Therefore, the

binding and adaptation of this portion of SsCU-NH
to the active site of the protease could be facilitated by
the concerted motion of these two segments. Neverthe-
less, because we were unable to isolate the proteolytic
fragment of 10.6 kDa, which was completely digested
by the proteases, we cannot exclude the possibility that
a first proteolytic cleavage could occur in loop 275–
288, which is a flexible and exposed loop protruding
towards the exterior of each monomer and, subse-
quently, the digestion was prolonged until segment
228–238.
Residues involved in Ca
2+
-coordination and in
substrate binding are shown in Fig. 5A. Residues D9,
D14, I121 and D238 participate in Ca
2+
coordina-
tion. These residues, with the exception of I121,
which coordinates the ion with the oxygen of its main
chain, are strictly conserved in the NH family
(Fig. 1), and are almost perfectly superimposed on
the structures of SsCU-NH and of the templates
(Fig. 5B). Residues D13, N37, N156, E162 and N164,
and again D238, are able to form hydrogen bonds
with the oxygen moieties of the sugar. Furthermore,
these residues are strictly conserved in the NH family
as well as H79, which is near the oxygen O1¢ and is
considered to be one of the catalytic residues of the
protein [29,30]. Other neighboring residues of ribose

probably form the wall of the active site for pyrimi-
dine binding. I157 and F163 are two hydrophobic
residues that could interact with the hydrophobic
moiety of the pyrimidine ring, as well as Q229, which
replaces two more hydrophobic residues W232 and
Y231, respectively, in YbeK and YeiK.
Particular attention should be paid to H236, which
is differently positioned with respect to the correspond-
ing residue in YbeK and YeiK (Fig. 1). The presence
of a P237 residue between H236 and D238 forces
H236 to go farther from the active site, with P237
superimposed on H239 of YeiK and H240 of YbeK
(Fig. 5B), which are considered to be involved in the
catalytic mechanism. In particular, this residue was
considered as a putative proton donor to the N3 or
A
AB
B
C
D
H10
H11
H13
H1
H2
C-ter
H12
H9
H7
H8

H6H5
H4
H3
S7
S8
S10
S11
S9
S6
S5
S4
S1
S2
S3
N-ter
Fig. 4. 3D structure of SsCU-NH. (A) Tetrameric assembly of SsCU-NH (cyan) compared to the template YeiK (yellow). The Ca ions in the
active site of YeiK are represented as orange spheres. Capital letters indicate the monomers. (B) Structure of the monomer. Helices are rep-
resented as red cylinders and b-strands as yellow arrows. Secondary structures are labelled with a progressive number, from N- to C-end.
The arrow indicates the putative site of cleavage by proteases (see text).
CU-NH from S. solfataricus M. Porcelli et al.
1906 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
O2 atoms in the hydrolysis of uridine [29,30]. How-
ever, mutagenic studies showed that H239A mutant of
YeiK has an increased K
m
but an unchanged k
cat
with
respect to the wild-type enzyme, therefore suggesting a
role for H239 in substrate binding but not in direct

proton transfer and catalysis [29]. The observation that
this residue is able to influence the affinity of the
enzyme for the substrate could explain why the affinity
of SsCU-NH for its substrates is lower than that of
homologous enzymes from E. coli.
Previous work in the area of understanding the
structural mechanisms of protein stability has identi-
fied some common features of thermophilic proteins
and has demonstrated that, generally, the stability of
thermophilic proteins is due to a combination of sev-
eral structural concurrent factors [40–42]. It has also
been reported that some thermophilic proteins employ
higher states of oligomerization to improve their
thermostability. Because SsCU-NH exists as a homo-
tetramer, additional criteria relating to the tetramer
interface (size, shape, inter-subunit hydrogen bonds
and salt bridges, and bridging solvent molecules) could
be also evaluated.
The extreme thermostability of SsCU-NH has also
generated much interest. In Table 2, we compare the
3D model of SsCU-NH with that of Ec-YeiK to iden-
tify structural features that might result in thermo-
stability. The comparison is complicated by the low
sequence identity, making it difficult to determine
which of the many residue changes contributes most
significantly to the increased stability of SsCU-NH.
F163
V78
N37
D9

H79
D14
D238
D13
H236
I121
P237
N164
E162
N156
Q229
Ca
Ribose
F163
V78
N37
D9
H79
D14
D238
D13
H236
I121
P237
N164
E162
N156
Q229
Ca
Ribose

A
B
Fig. 5. Active site of SsCU-NH. (A) Resi-
dues participating in Ca and ribose binding
and those predicted to participate in nucleo-
side binding are represented in stick mode,
with color code: carbon green, oxygen red,
nitrogen blue. Ribose is represented in ball
and stick mode, with the same color code.
Ca is represented as a sphere colored in
magenta. (B) Superposition of the residues
in the active site of YeiK (cyan) and YbeK
(yellow) to the residues in the active site of
SsCU-NH (colored by atom type code).
Ribose is represented in ball and stick mode
and Ca is represented as a sphere colored
in magenta. The figure is in stereo mode.
Table 2. Structural parameters of SsCU-NH and Ec-Yeik known to
affect protein thermostability.
SsCU-NH
Ec-YeiK
(1Q8F)
Secondary structure elements (%)
a
a-Helices 39.9 38.6
b-Strands 16.4 18.5
Other 20.4 20.4
Loops 23.3 22.5
b-Branched amino acids in helices (%)
b

18 25
Helix stability contributions (kcalÆmol
)1
)
c
22.3 31.5
Volume of cavities in monomer (A
˚
3
)
d
Buried 56 483
Surface 1220 1014
Total 1276 1497
Volume of cavities in tetramer (A
˚
3
)
d
Buried 867 2985
Surface 5672 3489
Total 6539 6474
Ile + Leu residues at monomers interface
A ⁄ B and C ⁄ D 1 Ile + 5 Leu 1 Ile
A ⁄ C and B ⁄ D 5 Ile + 1 Leu 2 Leu
a
Calculated using the program DSSP.
b
Calculated according to Fac-
chiano et al. [48].

c
Calculated according to Facchiano et al. [48].
d
Calculated using the program AVP.
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1907
The analysis of the composition and position of sec-
ondary structures shows that the model has a slightly
higher content in a-helices, a slightly lower content in
b structures, and a similar content in nonstructured
amino acids (coil) with respect to the template. Look-
ing at the model, these differences derive from longer,
rather than more, segments of secondary structure.
The structure of SsCU-NH is characterized by the
presence of Ile and Leu clusters, especially at the sub-
unit interfaces (Fig. 6A). In particular, the interfaces
between monomers A⁄ C and B ⁄ D are very rich in Ile
residues, with five Ile residues for each monomer
(I154, I157, I191, I261, I267) that form a group of
hydrophobic residues together with L273. Big Leu
clusters formed by five residues in each monomer
(L68, L69, L129, L132, L133), with the addition of
I174, are present at the interfaces between monomers
A ⁄ B and C⁄ D and act like a ‘hydrophobic zipper’ to
bring the two subunits together. Looking at the struc-
ture of YeiK (Fig. 6B), none of these hydrophobic
clusters are present in the quaternary structure,
although the number of Ile + Leu residues in each
monomer is similar (52 in YeiK, 56 in SsCU-NH).
We also analyzed the packing of the structure in

terms of presence of cavities in the interior of the pro-
tein. Using a probe of 0.5 A
˚
, we were able to calculate
the volume of buried and surface cavities for SsCU-
NH and the template, both in the monomer and in the
tetramer, and we found that the volume of buried cavi-
ties found in SsCU-NH is significantly lower than that
of YeiK (Table 2). This could be due to the higher
number of bulky residues (especially Trp, Tyr, Ile),
which are also generally shielded or partially shielded
from the solvent and therefore create a high compact
core in the structure of SsCU-NH. However, this result
should be interpreted with caution because it has been
reported that the estimation of packing density and
cavity volumes in homology models is intrinsically
noisy and may be inaccurate for the possible incorrect
modeling of nonconserved side chains between tem-
plate and target. This effect is dependent also on tem-
plate-target sequence identity [47].
A previous study [48] analyzed different factors that
concur together to stabilize helices in proteins. In the
present study, we applied the same analysis to our
model and to the template. The results obtained are
summarized in Table 2. Among the helix stabilizing
factors evaluated, the most significant one is the lower
content of b-branched residues in the helices of the
thermophilic protein (18% versus 25%). Indeed,
b-branched residues are known to destabilize helices.
Moreover, the evaluation of energetic contribution to

the protein stability indicates that, in SsCU-NH, the
helices contribute to the protein stability more than in
the mesophilic template.
Finally, it was previously noted that a single Cys
residue is present in SsCU-NH in a conserved position
with respect to the homologous NHs (Fig. 1). Our
model shows that this residue is deeply buried in the
interior of each monomer, completely inaccessible to
the solvent and therefore stabilized towards oxidation
at high temperatures.
Experimental procedures
Bacterial strains, plasmid, enzymes and
chemicals
Escherichia coli strain BL21(kDE3) was purchased from
Novagen (Darmstadt, Germany). Sulfolobus solfataricus
chromosomal DNA was kindly provided by C. Bertoldo
(Technical University, Hamburg-Harburg, Germany). Plas-
mid pET-22b(+) and the NucleoSpin Plasmid kit for
plasmid DNA preparation were obtained from Genenco
(Duren, Germany). Specifically synthesized oligodeoxyribo-
nucleotides were obtained from MWG-Biotech (Ebersberg,
Germany). Restriction endonucleases and DNA-modifying
enzymes were obtained from Takara Bio Inc. (Otsu, Shiga,
AB
Fig. 6. Ile + Leu clusters comparison. (A) Ile
(dark gray) and Leu (light gray) residues in
SsCU-NH. (B) Ile (dark gray) and Leu (light
gray) residues in YeiK. Backbone is repre-
sented as a ribbon and amino acids in CPK
mode.

CU-NH from S. solfataricus M. Porcelli et al.
1908 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
Japan). Pfu DNA polymerase was purchased from Strata-
gene (La Jolla, CA, USA). Thermolysin was obtained from
Boehringer (Mannheim, Germany). Trypsin, proteinase K,
subtilisin, nucleosides, purine and pyrimidine bases,
O-bromoacetyl-N-hydroxysuccinimide and standard pro-
teins used in molecular mass studies were obtained from
Sigma (St Louis, MO, USA). IPTG was from Applichem
(Darmstadt, Germany). Sephacryl S-200 and AH-Sepharose
4B were obtained from Amersham Pharmacia Biotech (Pis-
cataway, NJ, USA); poly(vinylidene fluoride) (PVDF)
membranes (0.45 mm pore size) were obtained from Milli-
pore (Bedford, MA, USA). All reagents were of the purest
commercial grade.
Enzyme assay
Nucleoside hydrolase activity was determined following the
formation of purine ⁄ pyrimidine base from the correspond-
ing nucleoside by HPLC using a Beckman system Gold
apparatus (Beckman Coulter Inc., Fullerton, CA, USA).
Unless otherwise stated, the standard incubation mixture
contained: 10 mmol Tris ⁄ HCl buffer (pH 7.4), 200 nmol of
the nucleoside and the enzyme in a final volume of 200 lL.
The incubation was performed in sealed glass vials for
5 min at 80 °C, except where indicated otherwise. The vials
were rapidly cooled in ice, and the reaction was stopped by
the addition of 100 lL of 10% trichloroacetic acid. Control
experiments in the absence of the enzyme were performed
to correct for nucleoside hydrolysis. When the assays were
carried out at temperatures above 80 °C, the reaction mix-

ture was preincubated for 2 min without the enzyme, which
was added immediately before starting the reaction. An
Ultrasphere ODS RP-18 column (Beckman) was employed
and the elution was carried out with 6 : 94 (v ⁄ v) mixture of
95% methanol and 0.1% trifluoroacetic acid in H
2
O. The
retention times of adenosine and adenine, inosine and hyp-
oxantine, guanosine and guanine, uridine and uracil, cyti-
dine and cytosine were 12.2 and 6.2 min, 10.5 and 4.7 min,
15.2 and 6.1 min, 6.8 and 4.2 min and 6.6 and 3.9 min
respectively. The amount of purine or pyrimidine base
formed is determined by integrating the peak of produced
nucleobase and converting this to the amount of nucleobase
by means of a standard curve (amount nucleobase versus
peak area). In all of the kinetic and purification studies, the
amounts of the protein was adjusted to ensure that no
more than 10% of the substrate was converted to product
and the reaction rate was strictly linear as a function of
time and protein concentration. One unit of enzyme activity
was defined as the amount of enzyme that catalyzes the
cleavage of 1 nmol of nucleosideÆmin
)1
at 80 °C.
Determination of kinetic constants
Homogeneous preparations of SsCU-NH were used for
kinetic studies. The purified enzyme gave a linear rate of
reaction for at least 10 min at 80 °C; thus, an incubation
time of 5 min was employed for kinetic experiments. All
enzyme reactions were performed in triplicate. Kinetic

parameters were determined from Lineweaver–Burk plots
of initial velocity data. K
m
and V
max
values were obtained
from linear regression analysis of data fitted to the Michael-
is–Menten equation. Values given are the mean ± SE from
at least three experiments. The k
cat
value was calculated by
dividing V
max
by the total enzyme concentration. Calcula-
tions of k
cat
were based on an enzyme molecular mass of
140 kDa.
Analytical methods for protein
Protein concentration was determined by the Bradford
method [49] using BSA as standard. Protein eluting from
the columns during purification was monitored at A
280
.
The concentration of purified SsCU-NH was estimated
spectrophotometrically using e
280
= 57870 m
)1
Æcm

)1
. The
molecular mass of the native protein was determined by
gel filtration at 20 °C on a calibrated Sephacryl S-200
column. The molecular mass under dissociating conditions
was determined at room temperature by SDS ⁄ PAGE, as
described by Weber et al. [50], using 12% or 15% acryl-
amide resolving gel and 5% acrylamide stacking gel.
Samples were heated at 100 °C for 5 min in 2% SDS
and 2% 2-mercaptoethanol and run in comparison with
molecular weight standards. Protein homogeneity was
assessed by SDS ⁄ PAGE. N-terminal sequence analysis of
the purified enzyme was performed by Edman degrada-
tion on a 473A sequencer (Applied Biosystems, Foster
City, CA, USA). Approximately 50 lg of purified pro-
tein, separated under denaturing conditions on a 15%
SDS ⁄ PAGE, was electroblotted onto a PVDF membranes
utilizing a Mini trans-blot transfer cell (Bio-Rad, Hercu-
les, CA, USA) apparatus, stained with Coomassie brillant
blue R-250 (0.1% in 50% methanol) for 5 min and
destained in 50% methanol and 10% acetic acid for
10 min at room temperature. Stained protein bands were
excised from the blot and their NH
2
-terminal sequences
were determined by automated Edman degradation on a
pulsed liquid sequencer (model 473A) connected online to
an HPLC apparatus for phenylthiohydantoin-derivative
identification, following the procedures suggested by the
manufacturer. The repetitive yield, based on stable amino

acids, was approximately 95%.
Stability and thermostability studies
The stability of SsCU-NH activity was examined at the
indicated temperatures. Immediately after the addition of
the compound, (time-zero control) and at different time
intervals, aliquots were removed from each sample and
analyzed for activity in the standard assay. Activity val-
ues are expressed as a percentage of the zero-time control
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1909
(100%). Enzyme thermostability was tested by incubating
the protein in sealed glass vials at temperatures in the
range 90–110 °C in an oil bath. Samples (2 lg) were
taken at time intervals and residual activity was deter-
mined by the standard assay at 80 °C. Activity values are
expressed as a percentage of the zero-time control
(100%).
Cell culture and homogenate preparation
Sulfolobus solfataricus was isolated from an acidic hot
spring in Agnano (Naples, Italy) and grown at 87 °Cas
previously reported [51]. For the homogenate preparation,
10 g of S. solfataricus cell paste was disrupted by sonication
in 10 mm Tris ⁄ HCl (pH 7.4). After centrifugation for 1 h
at 25 000 g, the supernatant was extensively dialyzed
against the same buffer.
Cloning and expression of the SsCU-NH-encoding
gene
The putative SsCU-NH gene SSO0505 (GenBankÔ acces-
sion number AE006681.1) from S. solfataricus was cloned
into the pET-22b(+) expression vector via two engineered

restriction sites (NdeI and EcoRI) introduced by PCR
with the primers 5¢-GCTATTGTGGTAGAATA
CATATG
AGACAC-3¢, sense, and 5¢-GGAGTTGTAAAAATTC
GAATTCTAAAGAGC-3¢, antisense (the introduced restric-
tion sites are underlined). Isolated genomic S. solfataricus
DNA (20 ng), hydrolyzed by EcoRI was used as a tem-
plate. PCR amplification was performed with Pyrococ-
cus furiosus DNA polymerase and a Minicycler (Genenco,
Duren, Germany) programmed for 30 cycles, each cycle
consisting of denaturation at 92 °C for 1 min, annealing at
55 °C for 2 min and extension at 68 °C for 2 min plus
5sÆcycle
)1
, followed by an extension final step of 15 min at
68 °C. The amplified gene (25 ng), hydrolyzed by NdeI and
EcoRI was inserted into pET22b(+) (150 ng) cut with the
same restriction enzymes. The recombinant plasmid was
named pET-SsCU-NH. The nucleotide sequence of the
inserted gene was determined by MWG Biotech (Ebersberg,
Germany) to ensure that no mutations were present in the
gene.
For the expression of recombinant SsCU-NH, an over-
night culture of E. coli BL21(kDE3) cells transformed
with the plasmid pET-SsCU-NH was used as 0.5% inocu-
lum in 1 L of LB medium containing 100 lgÆmL
)1
ampi-
cillin at 37 °C. At a late stage of cellular growth (i.e.
when the culture reached A

600
of 3.0), IPTG was added
at 1 mm final concentration and the induction was pro-
longed for 16 h. Cells (10 g) were harvested by centrifu-
gation and lysed as described by Sambrook et al. [52].
The cell debris was removed by centrifugation at 20 000 g
for 60 min at 4 °C and the supernatant was used as a
cell extract.
Preparation of MTI-Sepharose
The preparation of MTI-Sepharose was performed as
described by Kim et al. [53] by treating AH-Sepharose 4B
(5 g) with 1 mmol of O-bromoacetyl-N-hydroxysuccinimide
and then coupling the resin with 10 mg of MTI. MTI was
prepared by enzymatic deamination of MTA utilizing non-
specific adenosine deaminase (adenosine aminohydrolase;
EC 3.5.4.4) purified from Aspergillus oryzae. MTA
(30 lmol) was incubated with adenosine deaminase at
37 °C for 16 h in Tris ⁄ HCl 0.1 m (pH 7.4) as previously
described [54] and the reaction was stopped by the addition
of 10% tricarboxylic acid. The formation of MTI was
checked by reverse-phase HPLC on a 4.6 · 250 mm Ultra-
sphere ODS (5 l m particle size) column (Beckman) using
an Agilent 1100 series cromatograph (Agilent Technologies,
Palo Alto, CA, USA). The column, was equilibrated and
eluted with a 20 : 80 (v ⁄ v) mixture of 95% methanol
and 0.1% trifluoroacetic acid in H
2
O at a flow rate of
1mLÆmin
)1

. The chromatogram showed that, after the
enzymatic reaction, the peak of MTA (retention time of
10 min) was replaced by a new peak, with a lower retention
time (7 min), thus indicating the complete conversion, in
these experimental conditions, of MTA to MTI.
Purification of recombinant SsCU-NH
All the purification steps were performed at room tempera-
ture. Recombinant SsCU-NH was purified in two steps.
The cell extract of BL21 E. coli cells expressing SsCU-NH
was heated at 100 °C for 10 min and centrifuged at
20 000 g for 60 min. After overnight dialysis against 10 mm
Tris ⁄ HCl (pH 7.4), the enzyme was applied to an affinity
column (2 · 12 cm) of MTI-Sepharose equilibrated with
20 mm Tris ⁄ HCl (pH 7.4). The column was washed step-
wise with 50 mL of the equilibration buffer and then with
the same buffer containing 0.3 m NaCl until A
280
reached
baseline. NH activity was then eluted with 20 mm Tris ⁄ HCl
(pH 7.4) containing 0.3 m NaCl and 3 mm inosine. Active
fractions were pooled, concentrated and dialyzed exten-
sively against 10 mm Tris ⁄ HCl (pH 7.4).
Limited proteolysis experiments
Limited proteolysis experiments were carried out by incu-
bating recombinant SsCU-NH with proteinase K, subtili-
sin, trypsin or thermolysin. Enzymatic digestion with
proteinase K, subtilisin or trypsin was performed in
10 mm Tris ⁄ HCl (pH 7.4) at 37 °C. The final mass ratio
of substrate protein to protease was 10 : 1. Hydrolysis
was stopped by the addition of phenylmethylsulfonyl fluo-

ride (final concentration 250 lm) and the samples were
assayed for SsCU-NH activity. Proteolytic inactivation of
SsCU-NH with thermolysin was carried out in the same
CU-NH from S. solfataricus M. Porcelli et al.
1910 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
substrate ⁄ protease final mass ratio in a solution of 20 mm
Tris ⁄ HCl (pH 7.4), containing 10 mm CaCl
2
at 60 °C.
At the desired time intervals, aliquots were removed,
the reaction was stopped by the addition of 5 mm final
concentration EDTA and samples were assayed for activ-
ity.
The degradation of the intact protein over 2 h of incuba-
tion was monitored by SDS ⁄ PAGE of the digested material
followed by staining with Coomassie blue R-250. For the
amino acid sequence analysis, samples of the digested
recombinant SsNH from SDS ⁄ PAGE analysis were electro-
phoretically blotted onto a PVDF membrane utilizing a
Bio-Rad Mini trans-blot transfer cell apparatus. Trans-
ferred proteins were stained with Coomassie blue (0.1% in
50% methanol) for 5 min, destained in 50% methanol and
10% acetic acid solution for 30 min at room temperature,
and allowed to air dry. Stained protein bands were excised
from the blot and their NH
2
-terminal sequences were
determined.
Multiple sequence alignment
Protein similarity searches were performed using the data

from Swiss-Prot and Protein Identification Resource data-
banks. The multiple alignment was constructed using the
program clustalw [55].
Modeling of the 3D structure of SsCU-NH
The structure of SsCU-NH was modeled using as refer-
ence the 3D crystallographic structures of two pyrimidine
nucleoside hydrolases from E. coli, namely YbeK (PDB
code: 1YOE) [30] and YeiK (PDB code: 1Q8F) [30]. These
two structures are those with the highest score and the
lowest E-value found with a BLAST [56] search against
the PDB [57] database. A further search for suitable tem-
plates was made with the servers SAM-T02 [58,59] and
FUGUE [60] and gave the same results, therefore confirm-
ing the correctness of the chosen templates. The sequences
of target and templates were aligned to each others and
to those of the first 30 nucleoside hydrolase proteins
found with a BLAST search towards the UniProt ⁄ Swiss-
Prot database [61] using the program clustal w [55]. The
PredictProtein server [62] was used to perform predictions
on type and position of secondary structure elements on
SsCU-NH, whereas the analysis of type and position of
secondary structure elements on the templates and on the
modeled structure of SsCU-NH was performed using dssp
[63]. A few additional manual adjustments to optimize the
position of the gaps, aiming to avoid their placement into
the segments of secondary structure, were finally
performed.
The program modeller, version 6.1 [64], as imple-
mented in the molecular simulation package insightii,
version 2000.1 (Accelrys Software Inc., San Diego, CA,

USA), and version 8.2 for Windows (available at: http://
salilab.org/modeller/about_modeller.html) was used to
model the monomeric structure of SsCU-NH. Five models
were created using each version of the program, setting
the highest level of optimization. The quality of the mod-
els and their stereochemical properties were analyzed using
the programs procheck [65] and prosaii [66], and the
best monomer, created by modeller, version 6.1, was
chosen for the creation of the tetrameric form of SsCU-
NH enzyme. The monomeric chains were superimposed
on the structure of YeiK, to maintain the same relative
orientation of the subunits, with the aid of insightii tools.
A mild optimization of the tetrameric structure was then
applied to reduce steric clashes, using 500 steps of the
Steepest Descent method, with a final gradient of
0.01 kcalÆmol
)1
. All atoms were allowed to relax with no
constraints. This procedure represents the best compromise
between the need for relieving steric clashes and the risk
of distorting the geometry of the protein with deep and
extensive minimization. This concept was previously
applied to other models of oligomeric proteins and com-
plexes [67].
The control of the final quality of all models was per-
formed again with procheck [65]. After the assembly and
the subsequent mild energy minimization applied for
reduction of steric clashes [67], the quality of the model in
terms of stereochemical parameters was slightly worse
(86.1% of residues in most favored regions of the Rama-

chandran plot); nevertheless, the number of residues in
disallowed regions was the same as for the nonminimized
structure.
Visualization and analysis of model features was carried
out using insightii. The solvent accessibility of the resi-
dues was calculated using the program naccess [68], by
rolling a probe atom of 1.40 A
˚
on the van der Waals sur-
face of the protein model. The analysis of cavities in the
proteins was made using the program avp [69], using a
probe of 0.5 A
˚
to assess the packing of the molecules.
The evaluation of helix stability was carried out according
to Facchiano et al. [48].
Docking of ribose and Ca
2+
into the binding site of
SsCU-NH was performed using, as a starting reference, the
position of the sugar and of the metal in the crystallo-
graphic template 1YOE. The two ligands were modelled in
each binding site of SsCU-NH, and then the structure of
the resulting complex was optimized to allow a better
accommodation of the ligands in their binding sites and to
decrease steric hindrance. Optimization was carried out
with 500 minimization steps with the Steepest Descent
method until the energy gradient reached the threshold
value requested by the default options (0.01 kcalÆmol
)1

).
Consistent valence force-field developed for insightii was
adopted to assign potentials and charges both to the pro-
tein and the ligand. Hydrogen bonds were calculated with
the tool Measure ⁄ H-bonds provided in insightii.
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1911
Acknowledgements
The authors thank Dr Susan Costantini, Laboratory
of Bioinformatics, ISA, Avellino, for providing a tool
for the calculation of the percentage of secondary
structures from a dssp file. This research was sup-
ported by a grant from ‘Regione Campania’ L.R.
n.5 ⁄ 2002 and was partially supported by the CNR-Bio-
informatics project.
References
1 Camici M, Tozzi MG, Allegrini S, Del Corso A, Sanfi-
lippo O, Daidone MG, De Marco C & Ipata PL (1990)
Purine salvage enzyme activities in normal and neoplas-
tic human tissues. Cancer Biochem Biophys 11, 201–209.
2 Schramm VL (1997) Enzymatic N-riboside scission in
RNA and RNA precursors. Curr Opin Chem Biol 1,
323–331.
3 Verse
´
es W & Steyaert J (2003) Catalysis by nucleoside
hydrolases. Curr Opin Struct Biol 13, 731–738.
4 Murzin AG, Brenner SE, Hubbard T & Chothia C
(1995) SCOP: a structural classification of proteins
database for the investigation of sequences and struc-

tures. J Mol Biol 247, 536–540.
5 Orengo CA, Michie AD, Jones S, Jones DT, Swindells
MB & Thornton JM (1997) CATH – a hierarchic classi-
fication of protein domain structures. Structure 5, 1093–
1108.
6 Miller RL, Sabourin CL, Krenitsky TA, Berens RL &
Marr JJ (1984) Nucleoside hydrolases from Trypano-
soma cruzi. J Biol Chem 259, 5073–5077.
7 Parkin DW, Horenstein BA, Abdulah DR, Estupinan B
& Schramm VL (1991) Nucleoside hydrolase from
Crithidia fasciculata. Metabolic role, purification, speci-
ficity, and kinetic mechanism. J Biol Chem 266, 20658–
20665.
8 Parkin DW (1996) Purine-specific nucleoside N-ribohy-
drolase from Trypanosoma brucei brucei. Purification,
specificity, and kinetic mechanism. J Biol Chem 271,
21713–21719.
9 Shi W, Schramm VL & Almo SC (1999) Nucleoside
hydrolase from Leishmania major. Cloning, expression,
catalytic properties, transition state inhibitors, and
the 2.5-A
˚
-crystal structure. J Biol Chem 274, 21114–
21120.
10 Cui L, Rajasekariah GR & Martin SK (2001) A non-
specific nucleoside hydrolase from Leishmania donovani:
implications for purine salvage by the parasite. Gene
280, 153–162.
11 Verse
´

es W, Decanniere K, Pelle
´
R, Depoorter J, Bro-
sens E, Parkin DW & Steyaert J (2001) Structure and
function of a novel purine specific nucleoside hydro-
lase from Trypanosoma vivax. J Mol Biol 307, 1363–
1379.
12 Janion C & Lovtrup S (1963) Pyrimidine nucleoside
hydrolase in Thermobacterium acidophilum. Acta Bio-
chim Pol 10, 183–189.
13 Petersen C & Møller LB (2001) The RihA, RihB, and
RihC ribonucleosidehydrolases of Escherichia coli. Sub-
strate specificity, gene expression, and regulation. J Biol
Chem 276, 884–894.
14 Ogawa J, Takeda S, Xie S-X, Hatanaka H, Ashikari T,
Amachi T & Shimizu S (2001) Purification, characteri-
sation, and gene cloning of purine nucleosidase from
Ochrobactrum anthropi. Appl Environ Microbiol 67,
1783–1787.
15 Magni G, Fioretti E, Ipata PL & Natalini P (1975)
Baker’s yeast uridine nucleosidase. Purification, compo-
sition, and physical and enzymatic properties. J Biol
Chem 250, 9–13.
16 Kurtz J-E, Exinger F, Erbs P & Jund R (2002) The
URH1 uridine ribohydrolase of Saccharomyces cerevisi-
ae. Curr Genet 41, 132–141.
17 Mitterbauer R, Karl T & Adam G (2002) Saccharomy-
ces cerevisiae URH1 (encoding uridine-cytidine N-ribo-
hydrolase): functional complementation by a nucleoside
hydrolase from a protozoan parasite and by a mamma-

lian uridine phosphorylase. Appl Environ Microbiol 68,
1336–1343.
18 Ribeiro JM & Valenzuela JG (2003) The salivary purine
nucleosidase of the mosquito Aedes aegypti. Insect
Biochem Mol Biol 33, 13–22.
19 Verse
´
es W, Van Holsbeke E, De Vos S, Decanniere K,
Zegers I & Steyaert J (2003) Cloning, preliminary
characterization and crystallization of nucleoside
hydrolases from Caenorhabditis elegans and Campylo-
bacter jejuni. Acta Crystallogr D Biol Crystallogr 59,
1087–1089.
20 Abusamhadneh E, McDonald NE & Kline PC (2000)
Isolation and characterization of adenosine nucleosidase
from yellow lupin (Lupinus luteus). Plant Sci 153, 25–
32.
21 Campos A, Rijo-Johansen MJ, Carneiro MF & Fevere-
iro P (2005) Purification and characterization of adeno-
sine nucleosidase from Coffea arabica young leaves.
Phytochemistry 66, 147–151.
22 Hassan FH & Coombs GH (1988) Purine and pyrimi-
dine metabolism in parasitic protozoa. FEMS Microbiol
Rev 54, 47–84.
23 Berens RL, Krug ED & Marr JJ (1995) Purine and
pyrimidine metabolism. In Biochemistry and Molecular
Biology of Parasites (Marr JJ & Mu
¨
ller M, eds), pp.
89–117. Academic Press, London.

24 Schramm VL (1998) Enzymatic transition states and
transition state analog design. Annu Rev Biochem 67,
693–720.
25 Schramm VL, Horenstein BA & Kline PC (1994) Tran-
sition state analysis and inhibitor design for enzymatic
reactions. J Biol Chem 269, 18259–18262.
CU-NH from S. solfataricus M. Porcelli et al.
1912 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
26 Degano M, Gopaul DN, Scapin G, Schramm VL &
Sacchettini JC (1996) Three-dimensional structure of
the inosine-uridine nucleoside N-ribohydrolase from
Crithidia fasciculata. Biochemistry 35, 5971–5981.
27 Verse
´
es W, Decanniere K, Van Holsbeke E, Devroede
N & Steyaert J (2002) Enzyme-substrate interactions in
the purine-specific nucleoside hydrolase from Trypano-
soma Vivax. J Biol Chem 277, 15938–15946.
28 Verse
´
es W, Barlow J & Steyaert J (2006) Transition-
state complex of the purine-specific nucleoside hydrolase
of T. vivax: enzyme conformational changes and impli-
cations for catalysis. J Mol Biol 359, 331–346.
29 Giabbai B & Degano M (2004) Crystal structure to 1.7
A
˚
of the Escherichia coli pyrimidine nucleoside hydro-
lase YeiK, a novel candidate for cancer gene therapy.
Structure 12, 739–749.

30 Muzzolini L, Versees W, Tornaghi P, Van Holsbeke E,
Steyaert J & Degano M (2006) New insights into the
mechanism of nucleoside hydrolases from the crystal
structure of the Escherichia coli YbeK protein bound to
the reaction product. Biochemistry 45, 773–782.
31 Estupin
˜
a
`
n B & Schramm VL (1994) Guanosine-inosine-
preferring nucleoside N-glycohydrolase from Crithidia
fasciculata. J Biol Chem 269, 23068–23073.
32 Parks RE Jr & Agarwal RP (1972) Purine nucleoside
phosphorylase. In The Enzymes, Vol. 7 (Boyer PD, ed.),
pp. 483–514. Academic Press, New York, NY.
33 Bzowska A, Kulikowska E & Shugar D (2000) Purine
nucleoside phosphorylases: properties, functions and
clinical aspects. Pharmacol Ther 88, 349–425.
34 Pugmire MJ & Ealick SE (2002) Structural analyses
reveal two distinct families of nucleoside phosphorylas-
es. Biochem J 361, 1–25.
35 Cacciapuoti G, Porcelli M, Bertoldo C, De Rosa M &
Zappia V (1994) Purification and characterization of
extremely thermophilic and thermostable 5¢-methylthio-
adenosine phosphorylase from the archaeon Sulfolobus
solfataricus. Purine nucleoside phosphorylase activity
and evidence for intersubunit disulfide bonds. J Biol
Chem 269, 24762–24769.
36 Appleby TC, Mathews II, Porcelli M, Cacciapuoti G &
Ealick SE (2001) Three-dimensional structure of a hy-

perthermophilic 5¢-deoxy-5¢-methylthioadenosine phos-
phorylase from Sulfolobus solfataricus. J Biol Chem 276,
39232–39242.
37 Cacciapuoti G, Forte S, Moretti MA, Brio A, Zappia V
& Porcelli M (2005) A novel hyperthermostable 5¢-
deoxy-5¢-methylthioadenosine phosphorylase from the
archaeon Sulfolobus solfataricus. FEBS J 272, 1886–
1899.
38 Zhang Y, Porcelli M, Cacciapuoti G & Ealick SE
(2006) The crystal structure of 5¢-deoxy-5¢-methylthio-
adenosine phosphorylase II from Sulfolobus solfataricus,
a thermophilic enzyme stabilized by intramolecular
disulfide bonds. J Mol Biol 357, 252–262.
39 Woese CR, Kandler O & Wheelis ML (1990) Towards
a natural system of organisms: proposal for the
domains archaea, bacteria, and eucarya. Proc Natl Acad
Sci USA 87, 4576–4579.
40 Mozhaev VV, Berezin IV & Martinek K (1988) Struc-
ture-stability relationship in proteins: fundamental tasks
and strategy for the development of stabilized enzyme
catalysts for biotechnology. CRC Crit Rev Biochem 23,
235–281.
41 Vieille C & Zeikus GJ (2001) Hyperthermophilic
enzymes: sources, uses, and molecular mechanisms for
thermostability. Microbiol Mol Biol Rev 65, 1–43.
42 Sterner R & Liebl W (2001) Thermophilic adaptation of
proteins. Crit Rev Biochem Mol Biol 36, 39–106.
43 Van der Oost J, Ciaramella M, Moracci M, Pisani FM,
Rossi M & de Vos WM (1998) Molecular biology of
hyperthermophilic Archaea. Adv Biochem Eng Biotech-

nol 61, 87–115.
43a She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard
G, Awayez MJ, Chan-Weiher CCY, Clausen IG, Curtis
BA, De Moors A et al. (2001) The complete genome of
the crenarcheon Sulfolobus solfataricus P2. Cell Biology,
doi:10.1073/pnas.141222098
44 Kampranis SC & Maxwell A (1998) Conformational
changes in DNA girase revealed by limited proteolysis.
J Biol Chem 273, 22606–22614.
45 Fontana A, Fassina G, Vita C, Dalzoppo D, Zamai M
& Zambonin M (1986) Correlation between sites of lim-
ited proteolysis and segmental mobility in thermolysin.
Biochemistry 25, 1847–1851.
46 Fontana A, Polverino de Laureto P, Spolaore B, Frare
E, Ricotti P & Zambonin M (2004) Probing protein
structure by limited proteolysis. Acta Biochim Pol 51,
299–321.
47 Chakravarty S, Wang L & Sanchez R (2005) Accuracy
of structure-derived properties in simple comparative
models of protein structures. Nucleic Acids Res 33, 244–
259.
48 Facchiano AM, Colonna G & Ragone R (1998) Helix
stabilizing factors and stabilization of thermophilic pro-
teins: an X-ray based study. Protein Eng 11, 753–760.
49 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal
Biochem 72, 248–254.
50 Weber K, Pringle JR & Osborn M (1972) Measurement
of molecular weights by electrophoresis on SDS-acryl-

amide gel. Methods Enzymol 26, 3–27.
51 De Rosa M, Gambacorta A, Nicolaus B, Giardina P,
Poerio E & Buonocore V (1984) Glucose metabolism in
the extreme thermoacidophilic archaebacterium Sulfolo-
bus solfataricus. Biochem J 224, 407–414.
52 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1913
53 Kim S, Nochumson S, Chin W & Paik WK (1978) A
rapid method for the purification of S-adenosylmethio-
nine: protein-carboxyl O-methyltransferase by affinity
chromatography. Anal Biochem 84, 415–422.
54 Schlenk F, Zydek-Cwick CR & Hutson NK (1971)
Enzymatic deamination of adenosine sulphur-com-
pounds. Arch Biochem Biophys 142, 144–149.
55 Thompson JD, Higgins DG & Gibson TJ (1994) clus-
tal w: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, posi-
tion-specific gap penalties and weight matrix choice.
Nucleic Acids Res 22, 4673–4680.
56 Altschul SF, Madden TL, Scha
¨
ffer AA, Zhang J,
Zhang Z, Miller W & Lipman DL (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein
database search programmes. Nucleic Acids Res 25,
3389–3402.
57 Berman H, Henrick K, Nakamura H & Markley JL

(2007) The worldwide Protein Data Bank (wwPDB):
ensuring a single, uniform archive of PDB data. Nucleic
Acids Res 35, D301–D303 (Database Issue).
58 Karplus K, Karchin R, Draper J, Casper J, Mandel-
Gutfreund Y, Diekhans M & Hughey R (2003) Com-
bining local-structure, fold-recognition, and new-fold
methods for protein structure prediction. Proteins
53(Suppl. 6), 491–496.
59 Karchin R, Cline M, Mandel-Gutfreund Y & Karplus
K (2003) Hidden Markov models that use predicted
local structure for fold recognition: alphabets of back-
bone geometry. Proteins 51, 504–514.
60 Shi J, Blundell TL & Mizuguchi K (2001) FUGUE:
sequence-structure homology recognition using environ-
ment-specific substitution tables and structure-depen-
dent gap penalties. J Mol Biol 310, 243–257.
61 The UniProt Consortium (2007) The universal protein
resource (UniProt). Nucleic Acids Res 35, D193–D197
(Database Issue).
62 Rost B, Yachdav G & Liu J (2004) The PredictProtein
Server. Nucleic Acids Res 32, W321–W326 (Web Server
issue).
63 Kabsch W & Sander C (1983) Dictionary of protein
secondary structure: pattern recognition of hydrogen-
bonded and geometrical features. Biopolymers 22, 2577–
2637.
64 Sali A & Blundell TL (1993) Comparative protein mod-
elling by satisfaction of spatial restraints. J Mol Biol
234, 779–815.
65 Laskowski RA, MacArthur MW, Moss DS & Thornton

JM (1993) PROCHECK: a programme to check the ste-
reochemical quality of protein structures. J Applied
Crystallogr 26, 283–291.
66 Sippl M (1993) Recognition of errors in three-dimen-
sional structures of proteins. Proteins 17, 355–
362.
67 Marabotti A & Facchiano AM (2005) Homology mod-
eling studies on human galactose-1-phosphate uridylyl-
transferase and on its galactosemia-related mutant
Q188R provide an explanation of molecular effects of
the mutation on homo- and heterodimers. J Med Chem
48, 773–779.
68 Hubbard SJ, Campbell SF & Thornton JM (1991)
Molecular recognition. Conformational analysis of lim-
ited proteolytic sites and serine proteinase protein inhib-
itors. J Mol Biol 220, 507–530.
69 Cuff AL & Martin AC (2004) Analysis of void vol-
umes in proteins and application to stability of the
p53 tumour suppressor protein. J Mol Biol 344, 1199–
1209.
CU-NH from S. solfataricus M. Porcelli et al.
1914 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS