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: 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 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 [...]... 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 templates was made with the servers SAM-T02 [58,59] and FUGUE [60] and gave the same results, therefore confirming 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. .. 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 Ca2+ 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 crystallographic template 1YOE The two ligands... 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 complexes [67] The control of the final quality of all models was performed 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 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 models 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 SsCUNH enzyme The monomeric... phosphorylases Biochem J 361, 1–2 5 35 Cacciapuoti G, Porcelli M, Bertoldo C, De Rosa M & Zappia V (1994) Purification and characterization of extremely thermophilic and thermostable 5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds J Biol Chem 269, 2476 2–2 4769 36 Appleby TC, Mathews II, Porcelli M,... using the data from Swiss-Prot and Protein Identification Resource databanks 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 reference 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... regions of the Ramachandran 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 residues was calculated using the program naccess [68], by ˚ rolling a probe atom of 1.40 A on the van der Waals surface of the protein model The analysis... a hyperthermophilic 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus J Biol Chem 276, 3923 2–3 9242 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, 188 6– 1899 38 Zhang Y, Porcelli M, Cacciapuoti G & Ealick SE (2006) The crystal... 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... potentials and charges both to the protein and the ligand Hydrogen bonds were calculated with the tool Measure ⁄ H-bonds provided in insightii FEBS Journal 275 (2008) 190 0–1 914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1911 CU-NH from S solfataricus M Porcelli et al Acknowledgements The authors thank Dr Susan Costantini, Laboratory of Bioinformatics, ISA, Avellino, for providing a tool for the calculation . Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus – biochemical characterization and homology modeling Marina. report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The