Báo cáo khoa học: A novel hyperthermostable 5¢-deoxy-5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus pdf

A novel hyperthermostable 5¢-deoxy-5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus Giovanna Cacciapuoti 1 , Sabrina Forte 1 , Maria Angela Moretti 2 , Assunta Brio 1 , Vincenzo Zappia 1 and Marina Porcelli 1 1 Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita ` di Napoli, Italy 2 Centro Regionale di Competenza in Biotecnologie Industriali (BioTekNet), Seconda Universita ` di Napoli, Italy 5¢-Deoxy-5¢-methylthioadenosine phosphorylase (MTAP) (EC 2.4.2.28) catalyzes the reversible phos- phorolysis to free adenine and 5-methylthioribose- 1-phosphate [1] of 5¢-deoxy-5¢-methylthioadenosine (MTA), a sulfur-containing nucleoside generated from S-adenosylmethionine. In eukaryotes, polyamine bio- synthesis represents the major pathway of MTA formation: two moles of MTA are released per mole of spermine and one mole of MTA per mole of sper- midine [2,3]. A phosphorolytic breakdown of the Keywords 5’-deoxy-5’-methylthioadenosine phosphorylase; disulfide bonds; hyperthermostability; purine nucleoside phosphorylase; Sulfolobus solfataricus Correspondence G. Cacciapuoti, Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita ` di Napoli, Via Costantinopoli 16, 80138, Napoli, Italy Fax: +39 081 5667519 Tel: +39 081 5667519 E-mail: giovanna.cacciapuoti@unina2.it (Received 14 January 2005, revised 16 February 2005, accepted 17 February 2005) doi:10.1111/j.1742-4658.2005.04619.x We report herein the first molecular characterization of 5¢-deoxy-5¢-methyl- thio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low K m (0.7 lm) for 5¢ -deoxy-5 ¢-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 °C and extremely thermostable with an apparent T m of 112 °C that increases in the presence of substrates. The enzyme is characterized by high kinetic sta- bility and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. More- over, the binding of 5¢-deoxy-5¢-methylthioadenosine induces a conforma- tional transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S ⁄ C261S and C262S show thermophilicity and thermostability fea- tures significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic ana- lysis with the homologous 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the align- ment of the protein sequence of SsMTAPII with those of SsMTAP and human 5¢-deoxy-5¢-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate. Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; IPTG, isopropyl-b- D-thiogalactoside; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus. 1886 FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS thioether is operative in Eukarya [1–4] and Archaea [5,6] while an hydrolytic cleavage of the molecule occurs in Bacteria [7] and plants [8]. MTA phosphorylase is a member of purine nucleoside phosporylases (PNPs), ubiquitous enzymes of purine metabolism that function in the salvage pathway [9]. PNPs belong to the recently defined nucleoside phos- phorylase-1 (NP-1) family [10]. This family includes enzymes with a single-domain subunit, The NP-1 family is further divided into two subfamilies on the basis of amino acid sequence homology, quaternary structure organization, and substrate specificity [10]. The homotrimeric PNPs, isolated from many mamma- lian tissues, are specific for the catalysis of 6-oxopurines and their nucleosides [9], while the homohexameric PNPs are the dominant form in Bacteria [9] and Arch- aea [5,6] and are characterized by a broad substrate specificity. They, in fact, accept both 6-oxo- and ⁄ or 6-aminopurines and their nucleosides as substrates. The two classes do not have sequence homology but the analysis of their three-dimensional structures showed significant similarity of their monomers [9,10]. To the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) belong enzymes which share a common two-domain subunit fold and a dimeric qua- ternary structure and are specific for pirimidine nucleo- sides [10]. MTA phosphorylase was first characterized in rat ventral prostate [1]. The enzyme was purified to homo- geneity from mammalian tissues [4,11,12] and from Sulfolobus solfataricus [5] and Pyrococcus furiosus [6]. Moreover, crystal structures have been obtained for human MTAP (hMTAP) [13] and for MTAP from S. solfataricus (SsMTAP) [14]. SsMTAP is a hexameric protein of 160 kDa made up of six identical subunits of 26.5 kDa. This enzyme shows a significant sequence identity to E. coli PNP. Like E. coli PNP [15], SsMTAP is able to cleave ino- sine, guanosine and adenosine; unique to SsMTAP, however, is the ability to recognize MTA as substrate [5]. On the other hand, like SsMTAP, 5¢-deoxy-5¢- methylthioadenosine phosphorylase from P. furiosus (PfMTAP) has a hexameric quaternary structure and a broad substrate specificity with 20-fold higher catalytic efficiency for MTA and adenosine than for inosine and guanosine [6]. Nevertheless, PfMTAP shows negli- gible sequence homology with SsMTAP while it shares about 50% identity with hMTAP, a trimeric enzyme with high substrate specificity for MTA [13]. SsMTAP and PfMTAP are both characterized by exceptionally high thermoactivity and thermostability with temperature optima and apparent melting tem- peratures above the boiling point of water. Three intersubunit disulfide bonds have been evidenced in the three-dimensional structure of SsMTAP [14] and two intrasubunit disulfides have been identified in PfMTAP [16]. Moreover, it has been demonstrated that these covalent links represent an important structural mech- anism adopted by these enzymes to reach superior levels of stability. SsMTAP and PfMTAP represent two of the very few intracellular proteins with disulfide bridges reported so far in the literature. It is well known, in fact, that disulfide bonds are a typical feature of secretory proteins [17] and because of the reductive chemical envi- ronment inside the cells [18] the presence of these cova- lent links in intracellular proteins from well known organisms, is limited to proteins involved in the mechanism of response to redox stress [19] or to pro- teins catalyzing oxidation–reduction processes [20]. Analysis of the complete genome sequence of S. sol- fataricus, a hypothetical 5¢-deoxy-5¢-methylthioadeno- sine phosphorylase, indicated as MTAPII, has been shown beside the well known SsMTAP. The observa- tion that this putative MTAP shares 50% identity with human MTAP allows us to hypothesize that MTAPII is a completely different enzyme from SsMTAP. To obtain structural information on MTAPII and to study the functional role played by this enzyme in the purine nucleoside phosphorylase pathway of S. solfa- taricus, we carried out the expression of the gene and the purification and the physicochemical characteri- zation of this novel MTAP from S. solfataricus (SsMTAPII). Moreover, to investigate on the presence of disulfide bonds and to obtain information on the role played by these covalent links in the enzyme ther- mostability we constructed by site-directed mutagenesis a single (Cys262Ser) and a double (Cys259Ser-Cys261- Ser) mutant in the C-terminal region of SsMTAPII. These mutants were expressed in E. coli and purified, and their activities and thermal properties were com- pared with those of the wild-type enzyme. Finally, from the comparative kinetic analysis we demonstrate that SsMTAPII is the first MTA-specific MTA phos- phorylase isolated so far from Archaea. Results and Discussion Cloning, primary sequence comparison, and expression The analysis of the complete sequenced genome of S. solfataricus revealed an open reading frame (SS02344) encoding a 270 amino acid protein homo- logous to human MTAP which is annotated as SsMTAPII. The putative molecular mass of the pro- tein predicted from the gene was 30.14 kDa and the G. Cacciapuoti et al. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS 1887 estimated isoelectric point was 6.54. To overproduce SsMTAPII the gene was amplified by PCR, as des- cribed in Experimental procedures and cloned into pET-22b(+) under the T 7 RNA polymerase promoter. Comparison of the deduced primary structure of SsMTAPII with enzymes present in GenBank Data Base indicated that the highest identity was with the hypothetical MTA phosphorylases from Sulfolobus tokodaii (77%), A. pernix (69%), Pyrococcus horikoshi (63%) and Pyrococcus abissi (62%). A high identity was found with the hypothetical MTAPII from P. furiosus (48%) and with the hypothetical PNP from Aquifex aeolicus (40%). Among the related enzymes isolated from various sources, SsMTAPII shows high sequence identity with PfMTAP (63%) and with hMTAP (51%) while no significant similarity was found with SsMTAP, the MTAP isolated from S. sol- fataricus [5]. As deduced from the gene, SsMTAPII contains seven cysteine residues per subunit. This evidence con- firms the current opinion that, in spite of their high sensitivity to the oxidation at high temperature [21], this residue is present in remarkable amounts in hyper- thermophilic proteins where it is probably involved in stabilizing disulfide bridges [14,16,22–25]. The recombinant SsMTAPII was expressed in sol- uble form in E. coli BL21 cells harboring the plasmid pET-SsMTAPII at 37 °C in the presence of IPTG. Under the experimental conditions selected for the expression of the enzyme, about 12 g of wet cell paste was obtained from 1 liter of culture. SDS ⁄ PAGE ana- lysis of cell-free extract of induced cells revealed an additional band of approximately 30 kDa which cor- responded 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 SsMTAPII production in E. coli BL21 cells harboring pET-SsMTAPII, was found to be of 0.52 unitsÆmg )1 at 70 °C, confirming that the SsmtapII gene had been cloned and expressed. Enzyme purification and properties SsMTAPII has been produced in a soluble form with levels of up to 8% of the total cell protein. Recombin- ant protein was easily purified to homogeneity 12.5- fold by a two-step purification procedure. The soluble fraction of the cell extract was heated at 100 °C for 10 min to eliminate considerable amounts of heat- labile host proteins. The remaining impurities were removed by an affinity chromatography on MTA- Sepharose. About 14 mg of enzyme preparation with a 57% yield was easily obtained from 1 L of culture. No processing occurred at the enzyme’s N-terminus in the E. coli system, as proven by sequence determination of the first 10 amino acid of SsMTAPII. In analogy with SsMTAP, thiol groups are not involved in the catalytic process, as SsMTAPII activity is not affected by alkylating, mercaptide-forming or oxidizing thiol reagents. It is interesting to note, in this respect, that human MTAP has an absolute require- ment for thiol-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds [4]. The purified enzyme was found to be homogeneous. SDS ⁄ PAGE of the final preparation revealed a single band with a molecular mass of 30 ± 1 kDa, which is consistent with the expected mass deduced from the primary amino acid sequence of the enzyme. Gel filtra- tion on an analytical column of Sephacryl S-200 sug- gests that, under native conditions, SsMTAPII forms a hexameric structure with a molecular mass of 180 ± 9 kDa. On the basis of these results SsMTAPII, like the homologous MTA phosphorylases purified from S. solfataricus and P. furiosus, belongs to the hexameric group of the NP-1 family even if, because of the high amino acid sequence identity, it is more similar to hMTAP, a typical member of homotrimeric PNPs. Thermophilicity, stability, thermostability and substrate protection The temperature dependence of SsMTAPII activity assayed in the range from 30 to 140 °C is reported in Fig. 1. The enzyme appears highly thermophilic; its Fig. 1. The effect of temperature on SsMTAPII 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 degrees Kelvin. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus G. Cacciapuoti et al. 1888 FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS activity increased sharply up to the optimal tempera- ture of 120 °C and a 50% activity was still observable at 133 °C. The Arrhenius plot shows a discontinuity at about 97 °C, with two different activation energies suggesting conformational changes in the protein struc- ture around this temperature. The stability of SsMTAPII to reversible denatura- tion was investigated by carrying out short time kinet- ics of thermal denaturation. The diagram of the residual activity after 5 min of preincubation as a func- tion of temperature, reported in Fig. 2A is character- ized by a sharp transition. From the corresponding plot (see inset) it is possible to calculate a transition temperature (apparent T m ) of 112 °C. To evaluate the possible stabilizing effect of substrates on the thermo- stability of SsMTAPII we measured the melting tem- perature of the enzyme in the presence of 100 mm phosphate or 5 mm MTA. As shown in Fig. 2A, both molecules exert a similar protection toward tempera- ture inactivation of the enzyme causing an increase of the apparent T m to 119 and 117 °C, respectively (see inset). This result indicates that the binding of these substrate raises the noteworthy conformational stabil- ity of the enzyme thus reducing its susceptibility to thermal denaturation. A similar substrate protection against thermal inactivation was observed for SsMTAP [5,26], PfMTAP [6] and for MTAP from human placenta [4]. To study the thermostability properties in terms of resistance to irreversible thermal inactivation, the residual activity of SsMTAPII after preincubation at temperatures between 80 and 115 °C was followed for up to 4 h (Fig. 2B). Thermal inactivation obeyed first order kinetics at all the temperatures tested. SsMTAPII displayed high thermostability with half-life of 22 min and 84 min at 115 and 105 °C, respectively. The activation energy (E a ) for SsMTAPII inactivation calculated from the Arrhenius plot (inset) was 7055 kJÆmol )1 , about sevenfold higher than the activa- tion energy calculated for the catalyzed reaction, indi- cating a notably high kinetic stability. Kinetic stability has been reported as a feature of some naturally occurring proteins that are trapped in their rigid native conformations by an energy barrier and therefore are resistant to unfolding. It has also been proposed that the resistance to SDS-induced denaturation is a common property of kinetically stable proteins and that it represents a probe for identifying this type of proteins [27]. Therefore, we verified this correlation in SsMTAPII. SsMTAPII resulted highly resistant to SDS-induced denaturation remaining com- pletely active up to 2% SDS concentration at room temperature, whereas it is significantly inactivated at 0 20 40 60 80 100 A B 80 90 100 110 120 130 Temperature (°C) Residual activity (%) -9 -8 -7 -6 250 260 270 1/T x10 5 ln K Tm 11 2 °C -9 -8 -7 250 255 260 265 1/T x10 5 Tm 11 9 °C -8 -7 -6 250 255 260 265 1/T x10 5 ln K Tm 117 ° C ln K 1 1.5 2 0 60 120 180 240 Time (min) Log of % activity -9 -8 250 260 270 280 1/T x10 5 ln K Fig. 2. Thermostability of SsMTAPII. (A) Residual SsMTAPII activity after 5 min of incubation at temperatures shown in the absence (s) or in the presence of 100 m M phosphate ( )or5mM MTA (m). Apparent T m values are reported in the inset. (B) Kinetics of ther- mal inactivation of SsMTAPII as a function of incubation time. The enzyme was incubated at 80 °C(d), 90 °C(4), 100 °C( ), 105 °C (m) and 115 °C(s) for the time indicated. Aliquots were then with- drawn and assayed for the activity as described under Experimental procedures. The derived Arrhenius plot is reported in the inset. G. Cacciapuoti et al. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS 1889 70 °C (Fig. 3A). The remarkable SDS-resistance of SsMTAPII suggests a structural rigidity of the protein and the occurrence of reduced local and global-unfold- ing transitions. Figure 3A also shows the protective effect exerted by 100 mm phosphate or 5 mm MTA on the stability of SsMTAPII in the presence of 2% SDS at 70 ° C. The binding of MTA is able to completely stabilize the enzyme that, after a 1-h incubation, remains fully active. On the other hand, a lower protec- tion (55% residual activity) is observed in the presence of phosphate. This protective effect indicates that both MTA and phosphate are able to form binary complexes with the enzyme suggesting the hypothesis that, in anal- ogy with human MTAP [28] also SsMTAPII could act via a random mechanism. On the contrary, very recently it has been demonstrated that the reaction catalyzed by MTAP from P. furiosus follows an ordered Bi-Bi mechanism with the phosphate binding preceding the nucleoside binding in the phosphorolytic direction [16]. On the basis of two kinds of evidence a similar ordered Bi-Bi mechanism could also be pro- posed for MTAP from S. solfataricus. In fact, crystal- lization experiments showed that this enzyme forms a binary complex with phosphate while MTA form only a ternary complex in cocrystallization with the enzyme and sulfate [14]. Furthermore, substrate-stabilization experiments showed that phosphate and ribose-1-phos- phate are able to protect SsMTAP against denaturation by SDS while MTA and adenine are not [26]. To fur- ther analyze the stability of SsMTAPII we carried out equilibrium transition studies by incubating the enzyme at increasing guanidinium chloride concentrations in 20 mm Tris HCl, pH 7.4 for 22 h at 25 °C. Fig. 3B shows the denaturation curve determined by monitor- ing the changes in fluorescence maximum wavelength upon excitation at 290 nm where only tryptophanyl residues are specifically excited [29]. The obtained de- naturation curve shows a single sigmoidal transition indicating an apparent two-state transition from the native to the unfolded state without any detectable intermediate. The 3.3 m guanidinium chloride value of the midpoint transition indicates that SsMTAPII is very resistant to chemical denaturation. To examine whether the guanidinium chloride- induced unfolding of SsMTAPII is reversible, the refolding reaction was induced by 20-fold dilution of the sample. Extensive dialysis was then carried out until the complete removal of the denaturant. The guanidinium chloride-induced unfolding proved to be reversible since the refolded enzyme, when assayed for catalytic activity, shows the same specific activity of the native form. Substrate-induced conformational changes The features of unusual stability of SsMTAPII against thermal inactivation and its notably high resistance to Fig. 3. (A) Effect of MTA and phosphate on the thermostability of SsMTAPII in the presence of 2% SDS. The enzyme was incubated at 70 °C with 2% SDS in the absence (s) and in the presence of 100 m M phosphate ( )or5mM MTA (m). At the time indicated, aliquots were withdrawn and assayed for MTA phosphorylase activ- ity as described under Experimental procedures. Activity values are expressed as percentage of the time-zero control (100%). (B) guan- idinium chloride-induced fluorescence changes in SsMTAPII. Fluor- escence changes are reported as k max by monitoring the shift in fluorescence maximum wavelength, in 20 m M Tris ⁄ HCl, pH 7.4. The spectrum was recorded at 25 °C after 22-h incubation. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus G. Cacciapuoti et al. 1890 FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS SDS- and guanidinium chloride-induced denaturation are indicative of a compact and rigid structure that allows protein to retain enzymatic function in the extreme experimental conditions. It has been reported that, in addition to SDS resistance, kinetic stability is correlated with resistance to proteolytic cleavage [27]. To verify this correlation in SsMTAPII and to obtain information about the flexible regions of the protein exposed to the solvent and susceptible to proteolytic attack, we subjected the enzyme to limited proteolysis. This technique is also useful for probing conformat- ional changes occurring in proteins after enzyme– substrate interaction. Recombinant SsMTAPII was subjected to proteo- lysis with three different proteases. Among these, trypsin was not able to cleave the enzyme while sub- tilisin and proteinase K produced essentially the same results, so only the results for one, proteinase K, are shown (Fig. 4). The time course for the hydrolysis of recombinant SsMTAPII with proteinase K (Fig. 4A) followed by SDS ⁄ PAGE (Fig. 4B) showed that a protein band with an apparent molecular mass of about 4.5 kDa less than that of SsMTAPII appears as the catalytic activity decreases. After 2-h incuba- tion the protein band becomes abundant (lane 4, Fig. 4B) and the activity drops to 25% (Fig. 4A). The proteolytic fragment was analyzed by Edman degradation. The analysis showed that the N-termi- nus was preserved, thus indicating that the proteo- lytic cleavage site is localized in the C-terminal region. These results indicate that, in analogy with human MTAP [13], the C-terminal peptide of Ss- MTAPII is necessary for the integrity of the active site. When the experiment was carried out in the presence of 5 mm MTA, the enzyme remained com- pletely active (Fig. 4A) and the proteolytic process did not occur (lane 6, Fig. 4B). A change in protein conformation can mask or uncover a cleavage site, and an alteration in the lim- ited proteolysis pattern of the protein can be indicative of conformational changes [30]. Thus, the change in the digestion pattern of SsMTAPII in the presence of MTA implies that this substrate binds to protein spe- cifically and induces a conformational change. This protection against proteolysis provides the first direct evidence of a MTA-induced conformational change in SsMTAPII. When SsMTAPII was subjected to limited proteo- lysis in the presence of 100 mm phosphate only a slight protection on the catalytic activity was observed at the early stage of incubation (Fig. 4A). Furthermore, the proteolytic pattern after 2-h incuba- tion remains almost unmodified (lane 5, Fig. 4B) indicating that phosphate does not bind SsMTAPII in its C-terminal region. The conclusions drawn from limited proteolysis experiments are strengthened by the analysis of the sequence alignment of SsMTAPII and hMTAP repor- ted in Fig. 5 that clearly shows that the C-terminal region of SsMTAPII contains, in well conserved posi- tions, the same residues that in hMTAP are involved in the binding with MTA [13] with the exception of Leu237 of hMTAP that is substituted in SsMTAPII with Thr229. 0 20 40 60 80 100 A B 0 20 40 60 80 100 120 Time (min) Residual activity (%) kDa M C 1 2 3 4 5 6 50 40 20 25 30 15 Fig. 4. Limited proteolysis of SsMTAPII with proteinase K and sub- strate protection. (A) Time course for the hydrolysis of SsMTAPII in the absence (s) and in the presence of 100 m M phosphate ( )or 5m M MTA (m). (B) SDS PAGE, lane M, molecular mass markers; lane C, SsMTAPII control; lanes 1–4, SsMTAPII (2 lg) after 15-, 30-, 60-, and 120-min incubation in the absence of substrates; lane 5, SsMTAPII after 2-h incubation in the presence of 100 m M phos- phate; lane 6, SsMTAPII after 2-h incubation in the presence of 5m M MTA. The final mass ratio of SsMTAPII to protease was 25 : 1. Aliquots of SsMTAPII at different incubation times at 37 °C with protease were taken from the reaction mixture and the hydro- lysis was stopped (see Experimental procedures). Samples were assayed for MTA phosphorylase activity at 70 °C, subjected to SDS ⁄ PAGE and gel stained with Coomassie brilliant blue. G. Cacciapuoti et al. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS 1891 Effect of reducing agents on enzyme stability The extreme resistance against the inactivation caused by temperature, SDS and guanidinium chloride and the occurrence of a so elevated number of cysteines, 42 in the overall hexamer, prompted us to hypothesize that SsMTAPII, in analogy with the homologous MTAP from S. solfataricus and P. furiosus contains disulfide bonds. This hypothesis is supported by the observation that five of seven cysteine residues per SsMTAPII subunit are well conserved in PfMTAP, where they are involved in these covalent links [6]. To test this hypothesis, we studied the effect of increasing concentrations of the disulfide-reducing agent dithio- threitol on SsMTAPII. The enzyme is fully stable after preincubation in the presence of dithiothreitol at extre- mely high concentrations (0.2, 0.4 and 0.8 m)upto 50 °C. Therefore we have performed thermostability studies in the presence of these levels of reducing agents at 80 °C. As shown in Fig. 6, at 80 °C the enzyme remains stable up to 0.4 m dithiothreitol whereas it becomes susceptible to the effect of the reducing agent as the concentration rises. At 0.8 m dithiothreitol, in fact, a remarkable loss of activity (30% residual activity) is observable after one hour incubation. This result offers convincing evidence of the presence of disulfide bonds and suggests a role played by these covalent links in the stabilization of the protein. The requirement of elevated tempera- tures and high concentrations of reducing agents to Fig. 5. Multiple sequence alignment of hMTAP, SsMTAPII and SsMTAP. The phosphate ( qw) ribose (m) and base (d) binding sites of hMTAP (above the sequence) and of SsMTAP (below the sequence) are indicated. Conserved residues between hMTAP and SsMTAPII are highlighted in a grey box. SsMTAPII cysteine residues are boxed. Fig. 6. Effect of reducing agents on SsMTAPII thermostability. The enzyme (2 lg) was incubated at 80 °C for different times in 20 m M Tris ⁄ HCl pH 7.4 in the absence (r) and in the presence of 0.4 M ( ), 0.6 M (m), and 0.8 M (d) dithiothreitol. At the time indicated, aliquots were withdrawn and assayed for MTA phosphorylase activ- ity as described under Experimental procedures. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus G. Cacciapuoti et al. 1892 FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS inactivate the enzyme suggests that the disulfide(s) being reduced is quite inaccessible. Effect of mutations on enzyme thermostability In the absence of the three-dimensional structure of SsMTAPII, actually under investigation, we utilized site-directed mutagenesis technique to obtain informa- tion on the cysteine residues involved in disulfide bonds. We preliminarily selected Cys259, Cys261 and Cys262 in the C-terminal region of the enzyme and prepared two mutants, C259S ⁄ C261S and C262S. We selected Cys259 and Cys261, as a disulfide (Cys246- Cys248) has been demonstrated in PfMTAP at the same conserved positions [16]. Moreover, Cys262 was chosen as a mutagenic target since it is positioned in a peculiar sequence CXCC. It has to be noted that these three cysteine residues are localized in the C-terminal region of the enzyme and that this region, as well as the N-terminal region, are usually highly flexible and disordered in mesophilic proteins and are thought to be the first position of the protein which undergoes denaturation at high temperature [31]. Therefore, the presence of disulfide bonds could increase the stability of these protein regions. Large-scale production of the two mutant proteins was performed as described above for the SsMTAPII. Purified mutant proteins showed, under either native (gel filtration) or denaturing (SDS ⁄ PAGE) conditions, M r values identical to wild-type SsMTAPII and proved to be fully active demonstrating that the substitutions were not disruptive. To compare the stabilities of the mutant and wild- type proteins in terms of thermoactivity and thermal denaturation we measured their optimal temperatures, apparent T m values and residual activities after 1-h incubation at 90°. As reported in Table 1, the substitu- tion of Cys259, Cys261 and Cys262 with Ser signifi- cantly affect the thermophilicity and thermostability of SsMTAPII. Both mutated forms showed the same optimum temperature that is 5 °C lower than the wild- type SsMTAPII. In contrast, they significantly differ in their thermostabilities. The double mutant, in fact, shows thermal properties lower than those of the single mutant, indicating a significant role of the pair C259- C261 in the stabilization of the protein. We also tested the effect of 0.4 m dithiothreitol on the stability of the double and single mutant at temper- atures where the two proteins exhibited full activity in the absence of the reducing agent. In agreement with the results of thermostability experiments, the mutant C259S ⁄ C261S is more susceptible than C262S to the reducing agent retaining 55% residual activity after 1-h incubation at 70 °C in comparison with 63% residual activity displayed by the single mutant after 1-h incuba- tion at 80 °C (data not shown). These results indicate that, in addition to C259, C261, and C262 other cys- teine residues in SsMTAPII are probably involved in disulfide bonds. The results also confirm the important stabilizing role of the pair C259-C261. The reduced thermostability properties of the two mutant proteins with respect to those of the wild-type enzyme and their higher susceptibility to reducing agents indicate that Cys259, Cys261 and Cys262 parti- cipate in the stabilization of the protein, probably forming disulfide bridges. The double mutant C259S ⁄ C261S deserves particular attention. The struc- tural CXC motif of this hypothetical disulfide is un- usual and only a few examples are reported in the literature including CSC in Mengo virus coat protein [32], CDC in Bacillus Ak.1 protease [33], CTC in chap- erone Hsp33 from E. coli [34], CGC in yeast thiol oxidase [35], and in MTAP from P. furiosus [16]. Recently, it has been demonstrated that a CGC motif in a mutant of E. coli thioredoxin reductase [36] dis- plays a disulfide reduction potential that is close to that of protein disulfide isomerase, the most efficient known catalyst of oxidative protein folding. These data indicate that the CXC motif is an efficient cata- lyst of disulfide isomerization and that it could play a crucial role in the oxidative protein folding. These con- siderations allow us to speculate that the CSC motif in SsMTAPII could also play a similar role in stabilizing the protein disulfide bridges against reductive damage. Comparative kinetic characterization of SsMTAP and SsMTAPII The occurrence in S. solfataricus of SsMTAPII, the second enzyme beside the already isolated and charac- terized SsMTAP devoted to MTA catabolism, promp- ted us to revaluate and define our knowledge about the biochemistry of MTAP in this archaeon. There- fore, with the aim of gaining insight into the physio- logical role of SsMTAPII and on its functional Table 1. Comparative stability features of SsMTAPII and its mutated forms. Optimum temperature (C°) Apparent T m (C°) Stability after 1 h at 90 °C (% of activity) SsMTAPII 120 112 100 C262S 115 106 68 C259S ⁄ C261S 115 102 37 G. Cacciapuoti et al. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS 1893 relationships with SsMTAP, we carried out a compar- ative kinetic analysis of the two enzymes. The K m and V max values for purine nucleoside substrates in the presence of saturating concentrations of phosphate were calculated and typical Michaelis–Menten kinetics were observed. Moreover, the relative efficiency of the nucleoside substrates was determined by comparing the respective k cat ⁄ K m ratios, which are the best meas- ure for comparison of the efficiency of product forma- tion and substrate preference. The kinetic parameters of SsMTAP and SsMTAPII are reported in Table 2. As shown in Table 2, SsMTAP is characterized by a broad substrate specificity that recognizes both 6-amino- and 6-oxo-purine nucleosides as substrates. The enzyme shows a higher affinity for adenosine (K m 25.4 lm) and inosine (K m 84 lm) compared to guano- sine (K m 113.6 lm) and MTA (K m 154.1 lm). Moreover, SsMTAP displays a lower catalytic efficiency with MTA (k cat ⁄ K m 13.9 · 10 4 s )1 Æm )1 ) than in the presence of other purine nucleosides, suggesting that it could more appropriately be considered a purine nucleoside phosphorylase. In contrast, inosine and guanosine are inactive as substrates of SsMTAPII suggesting a com- pletely different metabolic role for this enzyme. Like SsMTAP, SsMTAPII is able to recognize adenosine even if the values of affinity and catalytic efficiency for this substrate are about one order of magnitude lower than those of SsMTAP indicating that adenosine is not a physiological substrate of SsMTAPII. An interesting feature of SsMTAPII is the extremely high affinity for MTA with an apparent K m of 0.7 lm. This value is about 220-fold lower than that of SsMTAP and some more, about one order of magnitude lower than that of hMTAP (K m 5 lm) [4], making SsMTAPII the most MTA-specific enzyme among those reported in the literature. The comparison of the k cat ⁄ K m values for MTA of the two archaeal enzymes clearly indicates that SsMTAPII is the enzyme physiologically respon- sible in S. solfataricus of the catabolism of MTA. The results of substrate specificity studies are sup- ported by the analysis of the alignment of SsMTAPII sequence with those of SsMTAP and hMTAP repor- ted in Fig. 5, where the amino acid residues involved in the active site of the two enzymes are also indica- ted. As expected on the basis of the very high sequence identity between SsMTAPII and hMTAP, the amino acid residues at the active site of the human enzyme are well conserved in SsMTAPII with few substitutions that could be useful to justify why SsMTAPII, unlike hMTAP, is able to recognize adenosine as a substrate. We can first consider that in SsMTAPII the substi- tution of Val233 and Leu237 of the human enzyme with Ala225 and Thr229, respectively, and the absence of Leu279 could contribute to modify the hydrophobic environment near the 5¢-position of the purine nucleo- side allowing SsMTAPII to bind adenosine. The second observation comes from the evidence that Ile210 of hMTAP is replaced in SsMTAPII by Met204 that in turn is located at the conserved posi- tion of Met181 in SsMTAP sequence. It has been dem- onstrated that in SsMTAP this residue provides a key interaction with the ribose moiety. Moreover, a methi- onine residue equivalent to Met181 of SsMTAP has been found in all known purine nucleoside phosphory- lase structures [37–40]. Therefore, it can be speculated that the substitution of a isoleucine, a residue more suitable for the specific binding of the enzyme with the methylthio group of MTA, with a methionine could allow SsMTAPII, in analogy with SsMTAP, to recog- nize adenosine in addition to MTA. Finally, in SsMTAPII Ser16 and Ser91 replace Thr18 and Thr93, respectively, of human MTAP. The substitution of a threonine with a serine residue could make the ribose pocket environment less hydrophobic, thus allowing SsMTAPII to recognize adenosine. It is interesting to note that the two threonine residues of human MTAP, which specifically recognizes MTA, are replaced by serine residues in the structurally homo- logous human PNP [40] which instead requires a 5¢-hydroxyl group. On the basis of the reported results, SsMTAPII shows peculiar structural and functional properties. The enzyme, in fact, although characterized by the hexameric quaternary structure distinctive of bacterial PNP, exhibits the catalytic properties reminiscent with that of human enzyme. In addition, like the homolog- ous MTAP from S. solfataricus and P. furiosus, SsM- TAPII probably contains disulfide bonds. This observation strengthens the current hypothesis that intracellular hyperthermophilic proteins are stabilized by these covalent links. Table 2. Kinetic parameters of SsMTAP and SsMTAPII. Activities were determined at 70 °C as described in Experimental proce- dures. K m (lM) k cat (s )1 ) k cat ⁄ K m (s )1 ÆM )1 ) SsMTAP MTA 154.1 ± 8 21.4 ± 1 13.8 · 10 4 Adenosine 25.4 ± 1 43.6 ± 2 17.2 · 10 5 Inosine 84 ± 4 132 ± 7 15.7 · 10 5 Guanosine 113.6 ± 6 29.6 ± 2 26.1 · 10 4 SsMTAPII MTA 0.7 ± 0.03 15.4 ± 1 22 · 10 6 Adenosine 270.1 ± 13 107 ± 5 39.6 · 10 4 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus G. Cacciapuoti et al. 1894 FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS Experimental procedures Bacterial strains, plasmid, enzymes, and chemicals Plasmid pET-22b(+) and the NucleoSpin Plasmid kit for plasmid DNA preparation were obtained from Genenco (Duren, Germany). E. coli strain BL21(kDE3) was pur- chased from Novagen (Darmstadt, Germany). Specifically synthesized oligodeoxyribonucleotides were obtained from MWG-Biotech (Ebersberg, Germany) Restriction endonuc- leases and DNA-modifying enzymes were obtained from Takara Bio, Inc. (Otsu, Shiga, Japan). Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA, USA). [methyl- 14 C]AdoMet (50–60 mCiÆmmol )1 was supplied by the Radiochemical Centre (Amersham Bioscience, Bucking- hamshire, UK). MTA and 5¢-[methyl- 14 C]MTA were pre- pared from unlabeled and labeled AdoMet [41] and purified by HPLC [42]. Proteinase K, phenylmethylsulfonyl fluoride, O-bromoacetyl-N-hydroxysuccinimide and standard pro- teins used in molecular mass studies were obtained from Sigma (St. Louis, MO, USA). Guanidinium chloride, dithio- threitol and isopropyl-b-d-thiogalactoside (IPTG) were from Applichem (Darmstadt, Germany). Sephacryl S-200 and AH-Sepharose 4B were obtained from Amersham Pharma- cia Biotech, polyvinylidene fluoride membranes (0.45 mm pore size) were obtained from Millipore (Bedford, MA, USA). All reagents were of the purest commercial grade. Enzyme assay MTA phosphorylase activity was determined by measuring the formation of [methyl- 14 C]5-methylthioribose-1-phos- phate from 5¢-[methyl- 14 C]MTA [5]. Unless otherwise sta- ted, the standard incubation mixture contained the following: 20 lmol potassium phosphate buffer, pH 7.4, 80 nmol of [methyl- 14 C]MTA (6.5 · 10 5 c.p.m.Ælmol )1 ), and the enzyme protein in a final volume of 200 lL. The incubation was performed in sealed glass vials for 5 min at 70 °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% (v ⁄ v) trichloroacetic acid. The mixture was then applied to a Dowex 50-H + column (0.6 · 2 cm) equilibrated in H 2 0. 5-[methyl- 14 C]Methylthio- ribose-1-phosphate produced was eluted with 2.5 mL of 0.01 m HCl directly into scintillation vials and counted for radioactivity. Control experiments in the absence of the enzyme were performed in order to correct for MTA hydrolysis. When the assays were carried out at tempera- tures above 70 °C, the reaction mixture was preincubated for 2 min without the enzyme that was added immediately before starting the reaction. When inosine, guanosine, and adenosine were used as substrates, the formation of purine base was measured by HPLC using a Beckman system Gold apparatus. The amount of purine base formed is determined 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. An Ultrasil- CX column (Beckman) eluted with 0.05 m ammonium for- mate, pH 3 at a flow rate of 1 mLÆmin )1 was used when adenosine and ⁄ or guanosine were the substrates of the reac- tion. In these experimental conditions the retention times of adenosine and adenine, guanosine and guanine were 7.3 min and 12.4 min, and 4.2 min and 6 min, respectively. When the assays were carried out in the presence of inosine as sub- strate, 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 retention times of inosine and hypoxantine were 10.5 min and 4.7 min, respectively. The same HPLC assay has been carried out with unlabeled MTA as substrate. In this case an Ultrasphere ODS RP-18 column was equilibrated and eluted with 20 : 80 (v ⁄ v) mixture of 95% methanol and 0.1% trifluoroacetic acid in H 2 O. The retention times of MTA and adenine were 10 min and 4.2 min, respectively. 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. Determination of kinetic constants Homogeneous preparations of SsMTAPII and SsMTAP [5] were used for kinetic studies. The purified enzymes gave a linear rate of reaction for at least 10 min at 70 °C, thus, an incubation time of 5 min was than employed for kinetic experiments. All enzyme reactions were performed in tripli- cate. Kinetic parameters were determined for both enzymes by varying the concentrations of purine nucleosides in the assay mixture in the presence of 100 mm phosphate. 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 values were calculated by dividing V max by the total enzyme concentra- tion. Calculations of k cat were based on an enzyme molecular mass of 160 kDa for SsMTAP and 180 kDa for SsMTAPII. Analytical methods for protein Proteins were assayed by the Bradford method [43] using bovine serum albumin as standard. The molecular mass of the native protein was determined by gel filtration at 20 °C on a calibrated Sephacryl S-200 column (2.2 · 95 cm) equili- brated with 10 mm Tris ⁄ HCl, pH 7.4, containing 0.3 m G. Cacciapuoti et al. 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus FEBS Journal 272 (2005) 1886–1899 ª 2005 FEBS 1895 [...]... control) and at different time intervals, 25-lL aliquots were removed from each sample and analyzed for activity in the standard assay Enzyme thermostability was tested by incubating the protein in sealed glass vials at temperatures between 80 °C and 145 °C in an oil bath Samples (2 lg) were taken at time intervals and residual activity was determined by the standard assay at 70 °C Activity values are expressed... Cartenı` -Farina M, Gragnianiello V, Schettino MI & Zappia V (1986) Purification and characterization of 5¢-deoxy-5¢-methylthioadenosine phosphorylase from human placenta J Biol Chem 261, 12324–12329 5 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. .. of MTA-Sepharose (2 · 12 cm) prepared as described by Cacciapuoti et al [5] equilibrated with 20 mm Tris ⁄ HCl pH 7.4 The column was washed stepwise with 50 mL of the equilibration buffer and then with the same buffer containing 0.6 m NaCl until the absorbance at 280 nm reached the baseline MTA phosphorylase activity was then eluted with 20 mm Tris ⁄ HCl pH 7.4 containing 0.6 m NaCl and 3 mm MTA Active... the Clustal method [47] Site-directed mutagenesis The construct pET-SsMTAPII was used as a template for site-directed mutagenesis by the Quik-Change procedure (Stratagene) The primers: 5¢-CAGAAGAGGGGTCG AGTTCCAGTTGCAACAGTC-3¢ and 5¢-GACTGTTGC AACTGGAACTCGACCCCTCTTCTG-3¢ (nucleotide substitutions are underlined) were used to make two silent mutations which replaced Cys259 and Cys261 with Ser and the primers...5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus NaCl at a flow rate of 4 mLÆh)1 The column was calibrated by using standard proteins of known molecular mass The molecular mass under dissociating conditions was determined by SDS polyacrylamide gel electrophoresis at room temperature, as described by Weber et al [44] by using 12.5 or 15% acrylamide resolving gel and 5% acrylamide stacking... 5¢-GGTCGTGTTCCTGTAGCAACAGTCTG AAGACAG-3¢ (sense) and 5¢-CTGTCTTCAGACTGTT GCTACAGGAACACGACC-3¢ (antisense) were used to make one silent mutation which replaced Cys262 with Ser pET-SsMTAPII (100 ng) was used for PCR amplification The resulting PCR products were checked by DNA sequence analysis and then used to transform E coli BL21 (kDE3) for overproduction of the proteins utilizing the same protocol used for the. .. hydrolysis was stopped by the addition of phenylmethylsulfonyl fluoride (final concentration 250 lm) and the samples were assayed for MTA phosphorylase activity To follow the degradation of the intact protein over 2 h of incubation the digested material was submitted to SDS ⁄ PAGE followed by staining with Coomassie blue R-250 For the amino sequence analysis, samples of the digested recombinant SsMTAPII, after... expressed as a percentage of the zero-time control (100%) Cloning and expression of the SsMTAPIIencoding gene The putative SsmtapII gene (GenBankTM accession number AE006641) from S solfataricus was cloned into the pET22b(+) expression vector via two engineered restriction sites (NdeI and BamHI) introduced by PCR with the following primers 5¢-GTATTTCATCATATGATTGAGC-3¢ sense, and 5¢-CGTATACTATTGGATCCATTTG-3¢,... Bzowska A, Kulikowska E & Shugar D (2000) Purine nucleoside phosphorylases: properties, functions and clinical aspects Pharmacol Therapeutics 88, 349–425 1897 5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus 10 Pugmire MJ & Ealick SE (2002) Structural analysis reveals two distinct families of nucleoside phosphorylases Biochem J 361, 1–25 11 Della Ragione F, Oliva A, Gragnianiello... stained with Coomassie Blue or cut into thin slices that were assayed for MTA phosphorylase activity by incubating in the assay mixture at 70 °C for 10 min N-Terminal sequence analysis of the purified enzyme was performed by Edman degradation on an Applied Biosystem 473 A sequencer according to the manufacturer’s instructions The sample was subjected to SDS ⁄ PAGE and electroblotted on a poly(vinylidene) . A novel hyperthermostable 5¢-deoxy-5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus Giovanna Cacciapuoti 1 , Sabrina Forte 1 ,. than that of SsMTAPII appears as the catalytic activity decreases. After 2-h incuba- tion the protein band becomes abundant (lane 4, Fig. 4B) and the activity