Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus Giovanna Cacciapuoti 1 , Sabrina Gorassini 1 , Maria Fiorella Mazzeo 2 , Rosa Anna Siciliano 2 , Virginia Carbone 2 , Vincenzo Zappia 1 and Marina Porcelli 1 1 Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita ` di Napoli, Italy 2 Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides to produce ribose-1-phos- phate and a free purine base [1–3]. PNPs have been characterized in a variety of species and may be grouped into two main groups, PNP-1 and PNP-2. PNP-1 are found in prokaryotes, are homohexamers with a subunit of 26 kDa and recognize both 6-oxo and 6-amino purine nucleosides as substrates. PNP-2 are homotrimers, with a subunit molecular mass of Keywords CXC motif; 5¢-deoxy-5¢-methylthioadenosine phosphorylase; disulfide bonds; hyperthermostability; purine nucleoside phosphorylase Correspondence G. Cacciapuoti, Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita ` di Napoli, Via Costantinopoli 16, 80138, Napoli, Italy Fax ⁄ Tel: +39 081 5667519 E-mail: giovanna.cacciapuoti@unina2.it (Received 2 February 2007, revised 6 March 2007, accepted 12 March 2007) doi:10.1111/j.1742-4658.2007.05784.x We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5¢-deoxy-5¢-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family mem- bers. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfM- TAP indicates that only four residue changes are able to switch the specif- icity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 °C and is characterized by extreme thermodynamic stability (T m , 110 °C that increases to 120 °C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 °C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasub- unit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S ⁄ C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability. Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢- methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PfPNP, purine nucleoside phosphorylase from P. furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from S. solfataricus. 2482 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 30 kDa and accept only guanosine and inosine as substrates [3–5]. It is interesting to note that many organisms that express PNP-1 also express PNP-2 [5]. PNP is a ubiquitous enzyme of purine metabolism that functions in the salvage pathway of cells. In addi- tion to the intrinsic biochemical significance, PNP plays an important biomedical role. In fact, human PNP is a target for T-cell-related cancers and autoimmune dis- eases [6]. Moreover, differences in substrate specificity between Escherichia coli PNP and the human enzyme have been employed for the development of tumor- directed gene therapy [5,7–10]. In this strategy, tumor cells transfected with E. coli PNP gene are able to convert relatively nontoxic prodrugs into membrane- permeant cytotoxic compounds. To reduce the toxicity of prodrugs currently used with E. coli PNP, a good experimental approach could be the identification of PNPs with new substrate specificities. In this light, studies on the molecular and structural characterization of PNPs from hyperthermophilic Archaea could be useful to improve the tumor-directed gene therapy based on the activation of nucleoside analogs prodrugs. Hyperthermophilic Archaea are of extreme biotechno- logical interest not only for the exceptional stability of their biomolecules but also for the peculiar substrate specificity of their enzymes that provide unique models for studying and understanding enzyme evolution in terms of structure, specificity and catalytic properties [11–15]. In recent years, the increasing number of solved crystallographic structures has highlighted the presence of disulfide bonds in several hyperthermo- philic proteins [16–20], suggesting that disulfide bond formation represents a significant molecular strategy adopted by cytosolic hyperthermophilic proteins to reach higher levels of thermostability. In Archaea, three enzymes belonging to the PNP fam- ily have recently been isolated and characterized from the hyperthermophilic microorganisms Sulfolobus solfa- taricus (Ss) and Pyrococcus furiosus (Pf). These enzymes are classified as 5¢-deoxy-5¢-methylthioadenosine phos- phorylases, as they are able to catalyze the phosphoroly- tic cleavage of 5¢-deoxy-5¢-methylthioadenosine (MTA), a natural sulfur-containing nucleoside formed from S-adenosylmethionine mainly through polyamine bio- synthesis [21,22]. The three enzymes, 5¢-deoxy-5¢-methyl- thioadenosine phosphorylase from S. solfataricus (SsMTAP), 5¢-deoxy-5¢-methylthioadenosine phosphor- ylase II from S. solfataricus (SsMTAPII) and 5¢-deoxy- 5¢-methylthioadenosine phosphorylase from P. furiosus (PfMTAP) show features of exceptional thermophilicity and thermostability with temperature optima and melting temperatures >100 °C [23–25] and are stabil- ized by disulfide bonds [16,20,26]. SsMTAP, which shows a significant sequence identity with E. coli PNP, is a hexamer consisting of six identical subunits of 26.5 kDa and utilizes inosine, guanosine, adenosine, and MTA as substrates [23]. The crystal structure of SsMTAP reveals that it contains three intermonomer disulfide bridges in each hexamer [16]. SsMTAP II is a homohexamer (subunit 30 kDa), characterized by extre- mely high affinity towards MTA. SsMTAPII shares 51% identity with human 5¢-deoxy-5¢ -methylthioadeno- sine phosphorylase (hMTAP) and is able to recognize adenosine [24] in contrast to hMTAP, which is highly specific for MTA. The crystal structure of SsMTAPII indicates a dimer of trimers with two pairs of intrasub- unit disulfide bridges [20]. Finally, PfMTAP is a hexa- meric protein that, like SsMTAPII, shares 50% identity with hMTAP. PfMTAP is characterized by a broad sub- strate specificity with 20-fold higher catalytic efficacy for adenosine and MTA than for inosine and guanosine [25]. PfMTAP is stabilized by two intrasubunit disulfide bridges [26]. The analysis of the complete genomic sequence of P. furiosus shows, beside PfMTAP, a second enzyme that, on the basis of the high identity with PfMTAP is annotated as MTAPII. We renamed this enzyme as PNP as it is completely unable to cleave MTA while, in analogy with human PNP, it is characterized by a strict substrate specificity towards inosine and guanosine. This paper describes the cloning, recombinant expression and structural and functional characteri- zation of purine nucleoside phosphorylase from the hyperthermophilic archaeon P. furiosus (PfPNP) aimed to elucidate the structure ⁄ function ⁄ stability relation- ship in this enzyme and to explore its biotechnological applications. By integrating classical biochemical meth- odologies with mass spectrometry, we assigned three intrasubunit disulfide bridges important for the enzyme stability. Finally, the characterization of the thermal properties of the C254S ⁄ C256S mutant allowed us to propose that the CXC motif in the C-terminal region of PfPNP may also account for the extreme thermo- stability of the enzyme. PfPNP, on the basis of its substrate specificity is the first example of a mamma- lian-like PNP reported in Archaea. Results and Discussion Analysis of PfPNP gene, primary sequence comparison and expression The analysis of the complete sequenced genome of P. furiosus revealed an open reading frame (PF0853) encoding a 265-amino acid protein homologous to hMTAP. This enzyme is annotated as hypothetical G. Cacciapuoti et al. Purine nucleoside phosphorylase from P. furiosus FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2483 MTAPII and has been renamed by us PfPNP. The putative molecular mass of the protein predicted from the gene was 29 208 Da. The coding region starts with an ATG triplet at the position 826577 of the P. furio- sus genome. The first stop codon TAG is encountered at the position 827374. Upstream from the coding region 24 bp before the starting codon there is a stretch of purine-rich nucleosides (CCTCC) that may function as the ribosome-binding site [27]. Putative promoter elements, which are in good agreement with the archaeal consensus [27] designed box A and box B are found close to the transcription start site. A hexa- nucleotide with the sequence TATTATA similar to the box A is located 19 bp upstream from the start codon and resembles the TATA box which is involved in binding the archaeal RNA polymerase [27]. A putative box B (ATGC) overlaps the ATG codon. Finally, a pyrimidine-rich region (TTTTTAT) strictly resembling the archaeal terminator signal [27], is localized 8 bp downstream from the translation stop codon. To overproduce PfPNP, 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 sequence [28] except for a single mutation at the third codon, where A was sub- stituted with G resulting in Arg instead of Gly. Since in repeated gene amplification experiments carried out utilizing different preparation of the same primers we always obtained the same result, it is possible to hypo- thesize that a mistake is present in GenBank at level of the third codon of PfPNP gene. Comparison of the deduced primary sequence of PfPNP with enzy- mes present in GenBank Data Base reveals a much higher similarity of PfPNP with members of MTAP family, such as MTAP from Pyrococcus abyssi (87% identity), MTAP from Pyrococcus horikoshii (84% identity), MTAP from Thermococcus kodakarensis (76% identity), than with members of PNP family such as PNP from Methanopyrus kandlery AV19 (51% iden- tity) and PNP from Aquifex aeolicus (47% identity). This evidence could also be noted by comparing the amino acid sequence of PfPNP with related enzymes characterized from various sources, that indicated a high sequence identity with PfMTAP (50%), SsMTAP- II (48%) and hMTAP (40%) while a lower identity was observed with E. coli PNPII (30%) and hPNP (27%). No significant similarity was found with E. coli PNP, SsMTAP, and PNP from Thermus thermophilus. The recombinant PfPNP was produced in a soluble form in E. coli BL21 cells harboring the plasmid pET- PfPNP at 37 °C in the presence of isopropyl-b-d-thio- galactoside. Under the experimental conditions selected for the expression, about 10 g of wet cell paste was obtained from 1 L of culture. The PfPNP activity of recombinant E. coli BL21 cells harboring pET-PfPNP, was 17.9 unitsÆmg )1 at 80 °C, confirming that PfPNP gene had been cloned and expressed. Enzyme purification and properties Recombinant PfPNP was purified to homogeneity by a fast and efficient two-step procedure that utilizes a heat treatment and affinity chromatography on MTI- Sepharose (Table 1). SDS⁄ PAGE of PfPNP reveals a single band with a molecular mass of 29 ± 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 which also revealed that the initial methionine was post-translationally removed. This result was con- firmed by MALDI-MS analysis of the HPLC purified protein. The experimental mass value (m ⁄ z 28 966.23) was in good agreement with the theoretical average molecular mass of the full length gene product without the N-terminal methionine (28 977.39 Da), being the observed mass difference partly due to the presence of disulfide bridges. The molecular mass of PfPNP was estimated to be 180 ± 9 kDa by size exclusion chromatography, which indicated a hexameric structure in solution. Therefore, on the basis of its quaternary structure PfPNP is a mem- ber of the hexameric group of PNPs (PNP-1) together with the structurally characterized PNPs from Archaea, including SsMTAP [16,23], SsMTAPII [20,24], and PfMTAP [25,26] and from Bacteria, such as PNP from E. coli (EcPNP) [29], PNP from T. thermophilus (TtPNP) [30], and E. coli uridine phosphorylase [31]. Substrate specificity and comparative kinetic characterization To elucidate the physiological role of PfPNP and its functional relationships with PfMTAP, we carried Table 1. Purification of recombinant purine nucleoside phosphory- lase from P. furiosus. A typical purification from 10 g of wet cells is shown. Total protein (mg) Total activity (units) Specific activity a (unitsÆmg )1 ) Yield (%) Purification (n-fold) Crude extract 134.0 126.9 0.95 100 1 Heat treatment 15.9 114.25 7.18 90.1 7.5 MTI-Sepharose 3.3 59.0 17.9 46.5 18.8 a Specific activity is expressed as nmol of hypoxanthine formed per min per mg of protein at 80 °C. Purine nucleoside phosphorylase from P. furiosus G. Cacciapuoti et al. 2484 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS out a detailed kinetic characterization of PfPNP and a comparative kinetic analysis of the two enzymes. Initial velocity studies carried out with increasing concentrations of purine nucleosides in the presence of saturating concentration of phosphate gave typical Michaelis–Menten kinetics. While PfMTAP showed a broad substrate specificity being able to phosphorolyti- cally cleave both 6-amino and 6-oxo purine nucleosides [25], PfPNP, in analogy with mammalian enzyme, is specific for guanosine and inosine with K m values of 122 and 322 lm, respectively. Moreover, the relative efficiency of the nucleoside substrates was determined by comparing the respective k cat ⁄ K m ratios. As shown in Table 2, the substrate activity of PfPNP with ino- sine and guanosine gave comparable k cat ⁄ K m values (2.61 · 10 7 and 2.2 · 10 7 , respectively) that are four orders of magnitude higher than those of PfMTAP for the same substrates, indicating that PfPNP is the enzyme physiologically involved in the 6-oxo-purine nucleoside catabolism in P. furiosus. When phosphate concentration was varied at fixed saturating concentra- tion of inosine, non-Michaelis–Menten kinetics were observed with two different K m values for phosphate of 6.2 and 259 lm. This result is in agreement with the data reported in the literature on the complexity of phosphate binding for PfMTAP [25] and for PNPs from various sources [32,33]. The results of substrate specificity studies are supported by the analysis of the sequence alignment of PfPNP, PfMTAP, hMTAP and hPNP reported in Fig. 1. The amino acid residues of PfPNP and Table 2. Kinetic parameters of PfPNP and PfMTAP. Activities were determined at 80 °C as described in Experimental procedures. K mapp (lM) k cat (s )1 ) k cat ⁄ K m app (s )1 ÆM )1 ) PfPNP Inosine 322 84.19 2.61 · 10 7 Guanosine 122 28.05 2.20 · 10 7 PfMTAP a MTA 147 24.46 1.66 · 10 5 Adenosine 109 22.79 2.09 · 10 5 Inosine 963 9.38 9.74 · 10 3 Guanosine 916 7.31 7.98 · 10 3 a The data for PfMTAP have already been published [25]. Fig. 1. Multiple sequence alignment of PfPNP, PfMTAP, hMTAP, and hPNP. The phosphate (w) ribose, (m) and base (d) binding sites of hMTAP (above the sequence) and of hPNP (below the sequence) are indicated. Identical residues between PfPNP and PfMTAP at the hypo- thetical active sites are highlighted in a grey box. PfPNP cysteine residues are shown in white lettering on a black background. G. Cacciapuoti et al. Purine nucleoside phosphorylase from P. furiosus FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2485 PfMTAP corresponding to those present at the active sites of hPNP [34] and hMTAP [35], respectively, were compared with highlight the changes that may account for the difference in substrate specificity among the two P. furiosus enzymes. As expected on the basis of the very high sequence identity (50%), the hypothetical active sites of PfPNP and PfMTAP are very similar and only few key residue changes are observable. Three important substitutions are localized at the level of the base binding site where Glu169, Asn211, and Ala213 of PfPNP replace Ser163, Asp204 and Asp206 of PfMTAP, respectively. It is important to note that these substitutions are exactly those that are respon- sible for the different substrate specificity of hPNP and hMTAP (Glu201, Asn243 and Val245 of hPNP instead of Ser178, Asp220 and Asp222 of hMTAP, respect- ively). The last important substitution is observable at the ribose pocket where His223 of PfPNP substitutes Ala215 of PfMTAP. Also in this case, the same substi- tution takes place in mammalian enzyme where the change of His257 of hPNP with Val233 of hMTAP makes hydrophilic the hydrophobic pocket, preventing the binding of the 5-methylthioribose moiety. As for the remaining differences between the hypothetical active sites of the two P. furiosus enzymes, they are all conservative substitutions except for the change of Ile56 of PfPNP with Phe57 of PfMTAP. It is interest- ing to note in this respect that the corresponding resi- due Tyr88 of hPNP is not determinant since the interactions between PNP and sugar ring are primarily hydrophobic [34]. In conclusion, only four substitu- tions are able to switch the specificity of the enzyme from 6-oxo to 6-amino purine nucleoside phosphory- lase still maintaining the same overall active site organ- ization. On the basis of the reported results, PfPNP shows peculiar structural and functional properties. The enzyme, in fact, although characterized by the hexameric quaternary structure distinctive of bacterial PNP, exhibits a substrate specificity that makes it the first archaeal mammalian-like PNP. Thermal properties and limited proteolysis The temperature dependence of the activity of PfPNP in the range from 30 °C to 140 °C is shown in Fig. 2. The enzyme is highly thermoactive; its activity increased sharply up to the optimal temperature of 120 °C and a 50% activity was still observed at 133 °C. This behavior led to a discontinuity in the Arrhenius plot at about 84 °C, with two different activation energies. To study the thermodynamic stability of PfPNP we measured the residual activity after 10 min incubation at increasing temperature. The corresponding diagram reported in Fig. 3A is characterized by a sharp trans- ition that allowed us to calculate an apparent melting temperature of 110 °C. This value increases to 120 °C in the presence of 100 mm phosphate indicating that this substrate is able to stabilize the enzyme toward temperature. A similar substrate protection against thermal denaturation was also observed for the homol- ogous enzymes SsMTAP [23], PfMTAP [25], SsMTAP- II [24], and hMTAP [36]. The resistance of PfPNP to irreversible heat inacti- vation processes was monitored by subjecting the enzyme to prolonged incubations in a temperature range from 100 to 115 °C and by measuring the resid- ual activity under standard conditions. As observed in Fig. 3B, the enzyme decay obeys first-order kinetics. The results obtained indicate that PfPNP is character- ized by a notably high kinetic stability retaining full activity after 4 h incubation at 100 °C (inset in Fig. 3B) and showing half-lives of 69, 12, and 5 min at 105, 110, and 115 °C, respectively. Kinetic stability has been reported as a property of some naturally occur- ring proteins that are trapped in their native conforma- tions by an high energy barrier that slows down the unfolding processes. It has also been reported in the literature that kinetically stable proteins are extremely resistant to SDS-induced denaturation [37]. Therefore, we incubated PfPNP in the presence of 2% SDS at increasing temperature and then we measured the cata- lytic activity under standard conditions. As shown in Fig. 4A, PfPNP remains fully active after 30 min 02 0 4 06 08 001 0510210906030 )C°( erutarepmeT Residual activity % 01 x T/ 1 5 2 3 4 5 053 003 052 log V Fig. 2. The effect of temperature on PfPNP activity. The activity observed at 120 °C is expressed as 100%. The assay was per- formed as indicated under Experimental procedures. Arrhenius plot is reported in the inset; T is measured in Kelvin. Purine nucleoside phosphorylase from P. furiosus G. Cacciapuoti et al. 2486 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS incubation at 50 °C and still retains 60% residual activity after 5 min incubation at 90 °C. Phosphate is able to increase the already high stability of PfPNP toward the detergent. In fact, after 15 min incubation at 100 °C with 2% SDS and 100 m m phosphate, the enzyme still shows about 20% residual activity (Fig. 4B) while in the same experimental conditions but in the absence of phosphate, it appears completely inactive. It is interesting to note that no protective effect against SDS inactivation has been observed in the presence of inosine indicating that only phosphate is able to form a binary complex with the enzyme. These results suggest that PfPNP, in analogy with PfMTAP [26], could act via an ordered Bi-Bi mechan- ism with the phosphate binding preceding the nucleo- side binding in the phosphorolytic direction. The high kinetic stability of PfPNP is indicative of a compact and rigid structure that allows the protein to retain its native state in extreme experimental condi- tions. It has been proposed that kinetic stability, by lim- iting the access of the protein to partially and globally unfolded conformations could be responsible not only for the extreme resistance to SDS-induced denaturation but also for the stability against proteolytic degradation [37]. To verify this hypothesis and to obtain information about the flexible regions of PfPNP exposed to the sol- vent and susceptible to proteolytic attack we subjected the enzyme to limited proteolysis. PfPNP resulted com- pletely resistant to several proteases, such as trypsin, chymotrypsin, proteinase K and subtilisin. Only ther- molysin was able to cleave the enzyme. Therefore, pro- teolytic degradation of PfPNP was investigated by measuring the residual activity after incubation with thermolysin at 60 °C followed by SDS ⁄ PAGE of the digested material. A protein band with an apparent molecular mass of about 2.6 kDa less than that of PfPNP appears as the proteolysis proceeds while no concomitant decrease of catalytic activity was observed. The analysis of the proteolytic fragment by Edman deg- radation showed that the amino terminus was preserved AB Fig. 3. Thermostability of PfPNP. (A) Resid- ual PfPNP activity after 5 min of incubation at temperatures shown in the absence (d) or in the presence of 100 m M phosphate (j). Apparent Tms are reported in the inset. (B) Kinetics of thermal inactivation of PfPNP as a function of incubation time. The enzyme was incubated at 100 °C (see inset), 105 °C(j), 110 °C(m), and 115 °C (d) for the time indicated. Aliquots were then withdrawn and assayed for the activity as described under Experimental proce- dures. Fig. 4. Effect of phosphate on the thermostability of PfPNP in the presence of 2% SDS. (A) The enzyme was incubated at 50 °C(s), 70 °C (m), 80 °C(j), and 90 °C(d) with 2% SDS. (B) The enzyme was incubated at 80 °C(j), 90 °C(d), and 100 °C(D) with 2% SDS in the presence of 100 m M phosphate. At the time indicated, aliquots were withdrawn and assayed for PfPNP activity as described under Experi- mental procedures. Activity values are expressed as percentage of the time-zero control (100%). G. Cacciapuoti et al. Purine nucleoside phosphorylase from P. furiosus FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2487 thus indicating that the proteolytic cleavage site is locali- zed in the C-terminal region. Moreover, the observation that no decrease of enzymatic activity occurred during proteolysis suggests that the C-terminal peptide of PfPNP is not necessary for the integrity of the active site. No substrate protection against proteolysis was observed, confirming the conclusions drawn from the analysis of the sequence alignment reported in Fig. 1 that highlights the absence of hypothetical substrate- binding sites in the C-terminal region of PfPNP. Effect of reducing agent and disulfide bond assignment In recent years, it has becoming evident that, in spite of their susceptibility to oxidative degradation, cysteine residues are abundant in genomes of various hyper- thermophilic Archaea and Bacteria [38]. Moreover, disulfide bonds are now known to occur in many hyperthermophilic and intracellular archaeal proteins [16–20], where they are thought to represent an important structural mechanism to obtain higher sta- bility. The unusual stability features of PfPNP and the elevated content of cysteine residues deduced from the gene (six per subunit) prompted us to investigate on the presence of stabilizing disulfide bonds. Therefore, the thermal stability of PfPNP was investigated by heating the enzyme in the presence of reducing agents. As reported in Fig. 5, after 1 h incubation at temper- atures until 70 °C, the enzyme remains completely stable even at high concentrations of dithiothreitol (0.8 m) whereas it becomes susceptible to the effect of the reducing agent as the temperature raises. In fact, in the presence of 0.4 m dithiothreitol, PfPNP retains only 20% activity after 1 h incubation at 100 °C. These results offer convincing evidence that PfPNP, in analogy with the homologous PfMTAP, contains disul- fide bonds important for the stability against thermal unfolding and denaturation. This hypothesis is suppor- ted by the observation that (a) five out of six cysteine residues of PfPNP are well conserved with respect to PfMTAP (Fig. 1), and (b) in PfMTAP four of these cysteine residues are involved in disulfide bonds [26]. To elucidate the S–S bridge arrangement, PfPNP was initially subjected to CNBr reaction and analyzed by MALDI-TOF-MS both in linear and in reflectron positive-ion mode. The signal at m ⁄ z 3761.25 generated from the C-terminal peptide 231–265 (monoisotopic molecular mass 3762.14 Da), occurred two mass units lower than expected on the basis of its amino acid sequence, thus indicating the presence of an intrapep- tide disulfide bond joining Cys254 and Cys256. More- over, the signal at m ⁄ z 13893.61 was assigned to a three peptides cluster, consisting of peptides 92–187 (average molecular mass 10838.37 Da), 188–201 (aver- age molecular mass 1555.87 Da) and 202–216 (average molecular mass 1499.81 Da) held together by two disulfde bonds (Table 3). In order to confirm the presence of the Cys254– Cys256 bridge, the peptide mixture originated from CNBr reaction was subjected to enzymatic digestion with Endoproteinase Glu-C. In the MALDI-TOF mass spectrum the signal at m ⁄ z 3160.80 corresponded to the peptide 236–265 containing the S–S bridge (mono- isotopic molecular mass 3159.81 Da). Nevertheless, isotope distribution of the signal could suggest the presence of a low percentage (10%) of the peptide having the cysteine residues in the reduced form (monoisotopic molecular mass 3161.80 Da), as can be deduced from the lower intensity of the peak at m ⁄ z 3161.83 and the higher intensity of peaks from m ⁄ z 3162.86 to m ⁄ z 3165.79 compared with the theor- etical isotope distribution expected for the peptide with the S–S bridge (Fig. 6). The S–S pattern of the other cysteine residues (136, 162, 190, 202) was determined cleaving the peptide chain between Cys136 and Cys162, by means of tryptic digestion of the protein. In the MALDI-TOF mass spectra the signal at m ⁄ z 3022.39 could be assigned to the pairing of the two peptides 158–167 (monoiso- topic molecular mass 1081.48 Da) and 179–197 02 04 06 08 001 8.06.04.02.00 [lotierhtoihtiD M] Residual activity(%) Fig. 5. Effect of reducing agents on PfPNP thermostability. The enzyme (2 lg) was incubated for 60 min in 20 m M Tris ⁄ HCl pH 7.4 containing dithiothreitol at indicated concentrations at 70 °C(d), 80 °C(j), 90 °C(m), and 100 °C(s). Aliquots were then withdrawn and assayed for PNP activity as described under Experimental procedures. Purine nucleoside phosphorylase from P. furiosus G. Cacciapuoti et al. 2488 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS (monoisotopic molecular mass 1942.00 Da) thus indi- cating that Cys162 is linked to Cys190. Similarly, the signal at m ⁄ z 4371.87 could be generated by the pep- tides 125–140 (average molecular mass 1923.17 Da) and 198–220 (average molecular mass 2450.87 Da) linked by a disulfide bond between Cys136 and Cys202 (Table 3). The S–S arrangement was further confirmed by submitting the tryptic peptide mixture to tandem mass spectrometric experiments. As an example, the MS ⁄ MS analysis of the peptide containing the S–S bond between Cys162 and Cys190 is reported in detail. The triply charged ion at m ⁄ z 1008.14, generated from disulfide-containing peptide (158–167) + (179–197), was selected for CID experiments and Fig. 7 reports the MS⁄ MS spectrum and the peptide amino acid sequence. Fragment ions belonging to series b (con- taining the N-terminal region of the peptide) and y (containing the C-terminal region) were originated from the entire sequence of both peptides 158–167 and 179–197. Diagnostic fragment ions of the S–S pairing resulted to be the singly charged ion y 7 (m ⁄ z 769.44) originated from the fragment 191–197 and its comple- mentary doubly charged ion b 12 (m ⁄ z 1127.50) origin- ated from the fragment 179–190 linked to the intact peptide 158–167. This is further demonstrated by the singly charged ion y 5 (m ⁄ z 559.27) produced from the fragment 163–167 and by the complementary doubly charged ion b 5 (m ⁄ z 1232.51) originated from the frag- ment 158–162 linked to the intact peptide 179–197. It is interesting to note that the disulfide bonds 136–202 and 254–256 are conserved in PfMTAP and SsMTAP- II confirming the disulfide arrangement of PfPNP. The presence of three disulfide bonds justify the extreme stability features of PfPNP. These covalent links, in fact, lowering the entropy of the unfolded poly- peptide and introducing at the same time new molecular relative intensity % z/ m 0 05 001 0713561306 1 3 relative intensity % z/m 68.2613 08.3613 38.1613 08.4613 97.5613 08.0613 0913 0413 0 05 001 n o it u birtsid epot os il a c i ter o e h T 1.6508 . 0 6 1 3 0010 8 .1613 9.7918.2613 6.861 8 .3613 1.8318.4613 7 . 711 8 .5613 noitu birtsid epotosilatnemirepxE 2 .3 5 0 8 .0 6 1 3 1. 6 838.16 1 3 00168.2613 7. 9 708.3613 9.8408.4613 2 .5 297.5613 z/m z/ m )%( y tisnetni e v italer )%(y tisne t ni evit al e r B A Fig. 6. Isotope distribution of the signal at m ⁄ z 3160.80 originated from the peptide 236–265 with a disulfide bridge. Experimen- tal (A) and theoretical (B) isotope distribu- tions are shown. Table 3. Disulfide arrangement of PfPNP. The solid lines indicate S–S bridges exactly assigned, while dashed lines refer to S–S bridges which could not be assigned in the experiment. Experimental m ⁄ z-values Amino acid sequence of disulfide-containing peptides Disulfide pattern obtained from CNBr reaction 3761.25 231 QKKSEDIVKLILAAIPLIPKERRCGCKDALKGATG 265 13893.61 92 KPGDFVILDQIIDFTVSRPRTFYDGEESPHERKFVAHVDFTEPY CPEIRKALITAARNLGLPYHPRGTYVCTEGPRFETAAEIRAYRILGGDVVGM 187 188 TQCPEAILARELEM 201 202 CYATVAIVTNYAAGM 216 Disulfide pattern obtained from tryptic digestion 3022.39 158 167 140 GTYVCTEGPR ILGGDVVGMTQCPEAILAR 197 4371.87 125 FVAHVDFTEPYCPEIR ELEMCYATVAIVTNYAAGMSGKK 220 G. Cacciapuoti et al. Purine nucleoside phosphorylase from P. furiosus FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2489 interactions into the protein structure could be respon- sible for increasing the kinetic stability that is in turn responsible for trapping the protein in its native state also in the extreme environmental conditions. Characterization of C254S ⁄ C256S mutant and role of the CXC motif To elucidate if the disulfide CGC localized at the C-terminus of PfPNP, in spite of its unusual structural features, could play a role in the stabilization of the protein we utilized site-directed mutagenesis to substi- tute Cys254 and Cys256 with serine. The large-scale preparation of the C254S ⁄ C256S mutant was per- formed as described above for recombinant PfPNP. Purified mutant protein showed, under either native (gel filtration) or denaturing (SDS ⁄ PAGE) conditions M r values identical to the wild-type PfPNP and proved to be fully active indicating the compatibility of the substitutions with the native state of the protein. We then carried out the characterization of the thermal properties of the mutant in comparison with those of PfPNP. The results obtained indicate that the substitu- tion of Cys254 and Cys256 with serine significantly affect both thermodynamic stability (T m , 102 °C) and kinetic stability (38% residual activity after 4 h incuba- tion at 100 °C, half-life of 35.5 min at 105 °C) of the enzyme suggesting an important role of the pair Cys254-Cys256 in the thermal stabilization of the enzyme. Disulfide bonds between cysteine residues separated by a single amino acid are extremely rare in nature. In addition to the disulfide CGC in PfMTAP [26] and CSC in SsMTAPII [24], the two highly PfPNP homol- ogous enzymes, only few examples are present in the literature [39–43]. The following considerations allowed us to hypothesize that the presence of a conserved unusual CXC disulfide in PfPNP, PfMTAP and SsM- TAPII would be not casual. Firstly, a CGC motif in a mutant of E. coli thioredoxin reductase [43] displays a disulfide reduction potential that is close to that of protein disulfide isomerase. This soluble eukaryotic protein is the most efficient known catalyst of the formation and isomerization of disulfide bonds [44], especially those within kinetically trapped, structured folding intermediates [45]. Second, a strict analogy may be observed between the CSC motif in SsMTAPII and the CGC motif in the thiol oxidase Erv2p from yeast, a FAD-dependent protein that can promote disulfide bond formation during the protein biosynthe- sis in the yeast endoplasmic reticulum [42]. In fact, as demonstrated by the elucidation of the three-dimen- sional structure, either in SsMTAPII [20] or in Erv2p [42] the CXC motif is part of a flexible C-terminal segment that can swing into the vicinity of another cysteine pair. In particular, in Erv2p the CGC motif was found to be involved in a disulfide relay that may help to shuttle electrons between dithiols of the sub- strate protein and the FAD-proximal disulfide [42]. Third, in analogy with Erv2p, the CGC motif of PfPNP is localized in the C-terminus of the enzyme that, as indicated by the protease sensitivity of the polypeptide chain at neighboring residues, is a flexible region. All these considerations and the results indica- ting a reduced thermodynamic and kinetic stability of the mutant C254S⁄ C256S with respect to the wild-type PfPNP, suggest that, as already hypothesized for SsM- TAPII [20,24], the two cysteines of the CGC motif in Fig. 7. MS ⁄ MS spectrum of the peptides 158–167 and 179–197 linked by S–S brid- ges. Diagnostic fragment ions b 5 and y 5 originated from the peptide 158–167, while ions b* and y* were from the peptide 179–197. Purine nucleoside phosphorylase from P. furiosus G. Cacciapuoti et al. 2490 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS PfPNP can undergo reversible oxidation-reduction to rescue the possible damage of the other two disulfide bonds. The presence of a low percentage of the protein with Cys254 and Cys256 in the reduced form further supports this hypothesis. It has been recently demonstrated that specific pro- tein disulfide oxidoreductases, structurally and functio- nally related to eukaryotic protein disulfide isomerase, play a key role in intracellular disulfide-shuffling in hyperthermophilic proteins [46–48]. In addition to protein disulfide oxidoreductases, the oxidized CXC motif in hyperthermophilic enzymes with intrasubunit disulfide bonds, such as PfPNP, PfMTAP, and SsM- TAPII, could represent an ingenious strategy adopted by these proteins to preserve their folded state in the extreme conditions. Experimental procedures Bacterial strains, plasmid, enzymes and chemicals MTA was prepared from AdoMet [23]. Thermolysin and Endoproteinase Glu-C were obtained from Boehringer (Mannheim, Germany). O-Bromoacetyl-N-hydroxysuccini- mide, cytochrome c, trypsin, cyanogen bromide (CNBr), angiotensin, adrenocorticotropic hormone fragment 18–39; nucleosides, purine bases and standard proteins used in molecular mass studies were obtained from Sigma (St Louis, MO, USA). Dithiothreitol and isopropyl-b -d- thiogalactoside were from Applichem (Darmstadt, Ger- many). Sephacryl S-200 and AH-Sepharose 4B were obtained from Amersham Pharmacia Biotech; polyvinyli- dene fluoride membranes (0.45 mm pore size) were obtained from Millipore (Bedford, MA, USA.). Specifically synthes- ized oligodeoxyribonucleotides were obtained from MWG- Biotech (Ebersberg, Germany). Plasmid pET-22b(+) and the NucleoSpin Plasmid kit for plasmid DNA preparation were obtained from Genenco (Duren, Germany). E. coli strain BL21(kDE3) was purchased from Novagen (Darms- tadt, Germany). P. furiosus chromosomal DNA was kindly provided by C. Bertoldo (Technical University, Hamburg- Harburg, Germany). Restriction endonucleases and DNA- modifying enzymes were obtained from Takara Bio, Inc. (Otsu, Shiga, Japan). Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA, USA). Nonspecific adeno- sine deaminase was purified 200-fold from Aspergillus oryzae powder (Sanzyme, Calbiochem, Los Angeles, CA, USA) according to Wolfenden et al. [49]. Enzyme assay Purine nucleoside phosphorylase activity was determined following the formation of purine base from the corres- ponding nucleoside by HPLC using a Beckman system Gold apparatus. The assay was carried out as already reported [25]. Unless otherwise stated, the standard incuba- tion mixture contained the following: 20 lmol potassium phosphate buffer, pH 7.4, 400 nmol of the nucleoside and the enzyme protein in a final volume of 200 lL. The incu- bation was performed in sealed glass vials for 5 min at 80 °C, except where indicated otherwise. Control experi- ments in the absence of the enzyme were performed in order to correct for nucleoside hydrolysis. When the assays were carried out at temperatures above 80 °C, the reaction mixture was preincubated for 2 min without the enzyme that was added immediately before starting the reaction. An Ultrasphere ODS RP-18 column was employed and the elution was carried out with 5 : 95 (v ⁄ v) mixture of 95% methanol and 0.1% trifluoroacetic acid in H 2 O. The retent- ion times of inosine and hypoxantine, guanosine and guan- ine were 10.5 min and 4.7 min, and 11.5 min and 4.3 min, respectively. The amount of purine base formed is deter- mined by measuring the percentage of the absorbance integrated peak area of purine base formed with respect to the total (nucleoside + purine base) absorbance integrated peak areas. In all of the kinetic and purification studies the amounts of the protein was adjusted so that no more than 10% of the substrate was converted to product and the reaction rate was strictly linear as a function of time and protein concentration. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the cleavage of 1 lmol of inosine per minute at 80 °C. Determination of kinetic constants Homogeneous preparations of PfPNP 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 Michaelis– Menten equation. Values given are the average from at least three experiments with standard errors. The k cat value was calculated by dividing V max by the total enzyme con- centration. Calculations of k cat were based on an enzyme molecular mass of 180 kDa. Analytical methods for protein Protein concentration was determined by means of the Bradford method [50] using bovine serum albumin as the standard. The molecular mass of the native protein was determined by gel filtration on a calibrated Sephacryl S-200 column as already reported [24]. The molecular mass under dissociating conditions was determined by SDS polyacryla- mide gel electrophoresis, as described by Weber et al. [51]. G. Cacciapuoti et al. Purine nucleoside phosphorylase from P. furiosus FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2491 [...]... Purification and characterization of 5¢-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus Substrate specificity and primary structure analysis Extremophiles 7, 159–168 26 Cacciapuoti G, Moretti MA, Forte S, Brio A, Camardella L, Zappia V & Porcelli M (2004) Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus Mechanism of the reaction and assignment of. .. electrophoresis and electroblotted on a polyvinylidene fluoride membrane prior to analysis Stability and thermostability studies The stability of PfPNP activity in the presence of SDS and dithiothreitol was examined at the indicated temperatures as reported in [24] Immediately after the addition of the compound (time-zero control) and at different time intervals, aliquots were removed from each sample and analyzed... in the standard assay Activity values are expressed as a percentage of the zero-time control (100%) Enzyme thermostability was tested by incubating the protein in sealed glass vials at temperatures between 100 °C and 115 °C in an oil bath Samples (2 lg) were taken at time intervals and residual activity was determined by the standard assay at 80 °C Cloning and expression of the PfPNP-encoding gene The. .. ternary complex of E coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, ˚ and phosphate (sulphate) at 2.1 A resolution J Mol Biol 280, 153–166 30 Tahirov TH, Inagaki E, Ohshima N, Kitao T, Kuroishi C, Ukita Y, Takio K, Kobayashi M, Kiramitsu S et al (2004) Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus J Mol Biol 337... Nature 365, 185–188 Purine nucleoside phosphorylase from P furiosus 46 Pedone E, Ren B, Ladenstein R, Rossi M & Bartolucci SF (2004) Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus A member of a novel protein family related to protein disulfide-isomerase Eur J Biochem 271, 3437–3448 47 Ladenstein R & Ren B (2006) Protein disulfides and protein disulfide... ⁄ prodrug therapy for pancreatic adenocarcinoma by E coli purine nucleoside phosphorylase and 6-methylpurine 2¢-deoxyriboside Pancreas 28, 54–64 FEBS Journal 274 (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS 2493 Purine nucleoside phosphorylase from P furiosus G Cacciapuoti et al 9 Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett LL & Parker WB (1994) Tumor cell bystander killing... (2007) 2482–2495 ª 2007 The Authors Journal compilation ª 2007 FEBS G Cacciapuoti et al Multiple sequence alignment Protein similarity searches were performed using the data from Swiss-Prot and Protein Identification Resource (PIR) data banks The multiple alignment was constructed using the clustal method [55] Purine nucleoside phosphorylase from P furiosus 18–39 (m ⁄ z 2465.1989) All the signals are singly... cromatograph The column, was equilibrated and eluted with a 20 : 80 (v ⁄ v) mixture of 95% methanol and 0.1% trifluoroacetic acid in H2O at a flow rate of 1 mLÆmin)1 The chromatogram showed that, after the enzymatic reaction, the peak of MTA (retention time 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... structure of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium J Mol Biol 337, 337–354, 32 Koellner G, Bzowska A, Wielgus-Kutrowska B, Luic M, Steiner T, Saenger W & Stepinski J (2002) Open and closed conformation of E coli purine nucleoside phosphorylase active center and implications... thermostable 5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus Purine nucleodide phosphorylase activity and evidence for intersubunit disulfide bonds J Biol Chem 269, 24762–24769 24 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 . here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The. Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus Giovanna