The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity.. SsCU-NH shares 34% sequence identity with pyrimidi
Trang 1archaeon Sulfolobus solfataricus – biochemical
characterization and homology modeling
Marina Porcelli1,2, Luigi Concilio1, Iolanda Peluso1, Anna Marabotti3, Angelo Facchiano3and
Giovanna Cacciapuoti1
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: marina.porcelli@unina2.it
(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 100C and is characterized by extreme thermodynamic stability (Tm= 106C) and kinetic stability (100% residual activity after 1 h incubation at 90C) 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.
Trang 2protozoa [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 87C,
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
Trang 3structures 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 bindcalcium-bind-ing 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.
Trang 4with 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 T7RNA
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 A600= 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)1Æmg)1
at 80C, 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)1Æmg)1at 80C
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 Ca2+-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 CaCl2 (data not shown), indi-cating that Ca2+ 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 Kmvalues of 310 lm and 970 lm
Trang 5respec-tively Moreover, as shown in Table 1, the relative
effi-ciency of these two substrates, determined by comparing
the respective kcat⁄ Kmratios, 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 40–130C 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 110C This behav-ior led to a discontinuity in the Arrhenius plot at approximately 80C, 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 106C The resistance of SsCU-NH to irreversible heat inactivation processes was monitored by subjecting the enzyme to prolonged incubations in the temperature range 90–110C 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 110C respectively
Table 1 Kinetic parameters of SsCu-NH Activities were
deter-mined at 80 C as described in the Experimental procedures.
K m app (l M ) k cat (s)1) k cat ⁄ K m app (s)1Æ M )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).
Trang 6To 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 37C or
with thermolysin at 60C 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 Ca2+-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.
Trang 7b-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 Ca2+-coordination and in substrate binding are shown in Fig 5A Residues D9, D14, I121 and D238 participate in Ca2+ 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
B
H10
H11 H13 H1 H2 C-ter
H12
H9
H6 H5 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).
Trang 8O2 atoms in the hydrolysis of uridine [29,30]
How-ever, mutagenic studies showed that H239A mutant of
YeiK has an increased Kmbut an unchanged kcatwith
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
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
Volume of cavities in tetramer (A˚3)d
Ile + Leu residues at monomers interface
a Calculated using the program DSSP b Calculated according to Fac-chiano et al [48]. cCalculated according to Facchiano et al [48].
d Calculated using the program AVP
Trang 9The 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 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 PlasPlas-mid 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,
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.
Trang 10Japan) 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 uscorrespond-ing 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 80C, except where indicated otherwise The vials
were rapidly cooled in ice, and the reaction was stopped by
the addition of 100 lL of 10% 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 80C, 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 H2O 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)1at 80C
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 80C; 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 Km and Vmaxvalues 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 kcat value was calculated by dividing Vmax by the total enzyme concentration Calcula-tions of kcat 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 A280 The concentration of purified SsCU-NH was estimated spectrophotometrically using e280= 57870 m)1Æcm)1 The molecular mass of the native protein was determined by gel filtration at 20C 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% acrylacryl-amide stacking gel Samples were heated at 100C 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 NH2-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