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• W2037487893 abstract "Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes. Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes. Among the fascinating properties of ribonucleotide reductases is their allosteric regulation of substrate specificity (general review in Ref. 1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar). These enzymes balance the reduction of ribonucleotides in a way that satisfies the cell's need for all four building blocks required for DNA synthesis. One protein catalyzes four separate reactions. The substrate specificity of the catalytic site for a given ribonucleotide is determined by binding of a specific deoxyribonucleoside triphosphate (dNTP) 1The abbreviations used are: dNTP, deoxynucleoside triphosphate; AMPPNP, 5′-adenylylimidodiphosphate; DTT, dithiothreitol or ATP to an allosteric site (substrate specificity site). Thus, binding of ATP or dATP induces activity toward pyrimidine ribonucleotides; binding of dTTP induces activity toward guanine ribonucleotides; and binding of dGTP induces activity toward adenine ribonucleotides. These effects are largely the same for all ribonucleotide reductases studied so far, except for some viral enzymes (2Nikas I. McLaugchlan J. Davison A.J. Taylor W.R. Clements J.B. Proteins Struct. Funct. Genet. 1986; 1: 376-384Crossref PubMed Scopus (46) Google Scholar). Many reductases have, in addition, a second allosteric site (activity site) that regulates their overall activity, with ATP promoting and dATP inhibiting enzyme activity (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar). Three classes of ribonucleotide reductases occur in nature. In addition to the earlier mentioned allosteric regulation of substrate specificity, all enzymes share a similar common free radical mechanism for the reduction of ribose and contain, for this purpose, a free radical as part of their protein structure. The three classes differ in the way in which the protein radical is produced and have evolved distinct protein structures (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar, 3Reichard P. Science. 1993; 260: 1773-1777Crossref PubMed Scopus (502) Google Scholar, 4Stubbe J. van der Donk W.A. Chem. Biol. 1995; 2: 793-801Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 5Sjöberg B.-M. Struct. Bonding. 1997; 88: 139-173Crossref Google Scholar). Class I reductases, with the aerobic Escherichia coli enzyme as the prototype, contain a tyrosyl free radical and have an α2β2-structure (6Fontecave M. Nordlund P. Eklund H. Reichard P. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 147-183PubMed Google Scholar). The large α2-dimer has been named the R1 protein (NrdA), and the small β2-dimer has been named the R2 protein (NrdB). The tyrosyl radical forms part of the R2 polypeptide, which also contains an oxygen-linked diferric center required for radical generation. This process requires oxygen, and class I enzymes function therefore only in aerobic organisms, both bacteria and eukaryotes. The R1 protein binds substrates and allosteric effectors and is the catalytic part of the enzyme. Class I has been divided into two subgroups (Ia (NrdAB) and Ib (NrdEF)) that differ from each other functionally and in their primary structures (7Jordan A. Pontis E. Åslund F. Hellman U. Gibert I. Reichard P. J. Biol. Chem. 1996; 271: 8779-8785Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In contrast to Ia, the class Ib reductases contain no allosteric activity site. The three-dimensional structures of complexes between the allosteric effector dTTP (specificity site) or the effector analog AMPPNP (activity site) and the R1 protein of the E. coli class Ia reductase have been solved, and the amino acid residues involved in the binding of each effector were identified (8Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The signum of class II reductases (NrdJ proteins) is their dependence on adenosylcobalamin, which acts as a radical generator and thus supplies the same function as the R2 protein of class I reductases. All class II reductases contain a single polypeptide chain, functionally related to the R1 protein of class I enzymes. They are widely spread among aerobic and anaerobic bacteria and do not depend on oxygen. The enzyme from Lactobacillus leichmannii (9Panagon D. Orr M.D. Dunstone J.R. Blakeley R.L. Biochemistry. 1972; 11: 2378-2388Crossref PubMed Scopus (57) Google Scholar, 10Booker S. Stubbe J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8352-8356Crossref PubMed Scopus (96) Google Scholar, 11Licht S. Gerfen G.J. Stubbe J. Science. 1996; 271: 477-481Crossref PubMed Scopus (283) Google Scholar) was, for a long time, the only well characterized member of this class and became its prototype. When, in recent years, the amino acid sequences of many more class II enzymes were determined, it became apparent that the L. leichmannii enzyme has an unusual sequence, different from those of most other class II as well as class I and III enzymes. Class III reductases (NrdDG proteins), with the anaerobic E. coli reductase as a prototype (12Reichard P. J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar), have again an α2β2-structure. The catalytic α-subunit carries not only both substrate and allosteric sites, but also the free radical required for catalysis, which, in class III reductases, is located on a glycyl residue at the C terminus of the polypeptide chain. The β-subunit generates this radical with the aid of an iron-sulfur cluster in an anaerobic reaction that requires reduced flavodoxin and S-adenosylmethionine (13Ollagnier S. Mulliez E. Gaillard J. Eliasson R. Fontecave M. Reichard P. J. Biol. Chem. 1996; 271: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The glycyl radical is oxygen-sensitive, and class III reductases can therefore operate only in strict anaerobic bacteria or facultative aerobic bacteria growing in the absence of oxygen. Within one class, the large α- and small β-subunits each share homologous amino acids. Between the classes, however, the α-subunits of the three prototypes for the classes show only very limited homology, suggesting no or only a distant evolutionary relation between the classes. However, recent studies of class II enzymes with deep evolutionary roots demonstrated that these enzymes and class I enzymes share homologous amino acids for effector binding at critical positions in the substrate specificity site (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar). As for class III, excepting the T4-induced enzyme (15Young P. Öhman M. Sjöberg B.-M. J. Biol. Chem. 1994; 269: 27815-27818Abstract Full Text PDF PubMed Google Scholar), the N termini of the α-peptides containing the hypothetical allosteric second dATP site show considerable homology to corresponding segments of some of the new class II enzymes as well as to that of the E. coli class I enzyme. The allosteric properties of several class I reductases (16Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 39-55Crossref PubMed Scopus (171) Google Scholar, 17Eliasson R. Pontis E. Jordan A. Reichard P. J. Biol. Chem. 1996; 271: 26582-26587Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and one class III reductase (18Eliasson R. Pontis E. Sun X. Reichard P. J. Biol. Chem. 1994; 269: 26052-26057Abstract Full Text PDF PubMed Google Scholar) have been investigated in detail by kinetic and effector binding experiments. Our knowledge of the allosteric behavior of class II enzymes is, however, incomplete. We know next to nothing concerning the recently described archaeal and deeply rooted eubacterial enzymes, and early studies of the L. leichmannii enzyme (19Beck W.S. J. Biol. Chem. 1967; 242: 3148-3158Abstract Full Text PDF PubMed Google Scholar, 20Chen A.K. Bhan A. Hopper S. Abrams R. Franzen J.S. Biochemistry. 1974; 13: 654-661Crossref PubMed Scopus (34) Google Scholar) need to be complemented in the light of recent knowledge. Here, we describe our experiments with three class II reductases: the previous prototype enzyme from L. leichmannii (9Panagon D. Orr M.D. Dunstone J.R. Blakeley R.L. Biochemistry. 1972; 11: 2378-2388Crossref PubMed Scopus (57) Google Scholar, 10Booker S. Stubbe J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8352-8356Crossref PubMed Scopus (96) Google Scholar), the archaeal thermophilic enzyme fromThermoplasma acidophilum (21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 53-58Crossref PubMed Scopus (50) Google Scholar), and the eubacterial hyperthermophilic enzyme from Thermotoga maritima (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar). BothT. acidophilum and T. maritima are deeply rooted organisms. L. leichmannii, instead, is a highly specialized microorganism, adapted to life in milk. The three enzymes had the opportunity to diverge extensively during evolution. Despite this, their substrate specificity was regulated in a similar manner, according to the same rules as for class I and III reductases. The thermophilic organisms showed a clear allosteric regulation only at elevated temperatures, suggesting that the proteins at lower temperature are not sufficiently flexible to transmit the conformational change resulting from effector binding to the catalytic site. None of the three enzymes was inhibited by dATP, and they all thus lack a functional activity site. However, the T. acidophilum reductase (but not the other two enzymes) meets the structural requirements for an activity site, and in effect, it did bind a second dATP. Our results will be discussed in light of a previously suggested model for the evolution of ribonucleotide reduction. Bacterial strains overproducing the L. leichmannii(10Booker S. Stubbe J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8352-8356Crossref PubMed Scopus (96) Google Scholar) and T. acidophilum (21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 53-58Crossref PubMed Scopus (50) Google Scholar) enzymes were kindly provided by Dr. J. Stubbe. The T. maritima ribonucleotide reductase was purified from an overproducing E. coli strain prepared earlier in this laboratory (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar). Labeled nucleoside di- and triphosphates were obtained from Amersham Pharmacia Biotech. They were diluted to the following specific activities: 14C-labeled substrates, 3–6 cpm/pmol; 3H-labeled substrates, 10–20 cpm/pmol; and 3H-labeled effectors, 200–450 cpm/pmol. Their radiopurity was checked by chromatography on polyethyleneimine strips. If necessary, they were purified by chromatography on DEAE-Sephadex with a volatile buffer. Small portions of the dissolved labeled nucleotides were stored at −80 °C. Batch cultures of bacteria were grown at 37 °C in Luria-Bertani broth with 100 μg/ml ampicillin to a finalA600 of 0.5, at which point isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.8 mm. The bacteria were incubated for another 3 h, harvested by centrifugation, and stored frozen. Except where indicated, all further procedures were done at close to 0 °C. Extracts of the bacteria were prepared as described earlier (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar) with the exception that streptomycin (at a final concentration of 1%) was included in the extraction buffer. This greatly facilitated centrifugation of the otherwise highly viscous extracts. The clear supernatant solutions were precipitated with solid ammonium sulfate to 50% saturation. After centrifugation, the precipitate was dissolved in buffer A (30 mm Tris-HCl, pH 7.5, and 2 mm DTT) and freed from ammonium sulfate by dialysis against the same buffer. The final solution contained ∼10 mg of protein/ml. This procedure was applied to 3 g of bacteria for the purification of the L. leichmannii enzyme and to 20–50 g of bacteria for the purification of the T. maritima and T. acidophilum enzymes. Overproduction of the enzyme was very efficient, and at this stage, we had a total of 83 mg of protein with a specific activity of 490 units/mg of protein. Half of this protein, dissolved in 2.6 ml of buffer A, was adsorbed onto a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) and eluted with a 0–1 m KCl gradient in buffer A. A peak of activity was recovered at 0.23 m KCl, and the central portion of this peak (16.5 mg of protein, specific activity = 750 units/mg of protein) was used for our experiments. After the ammonium sulfate precipitation of the protein from an extract of 20 g of bacteria, we recovered 1.0 g of protein with a specific activity of 14 units/mg of protein. The solution was heated at 55 °C for 30 min and centrifuged, and the clear supernatant solution was precipitated with solid ammonium sulfate to 50% saturation. The centrifuged precipitate was dissolved in 2 ml of buffer B (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, and 5 mm DTT) and freed from ammonium sulfate by dialysis against the same buffer. The enzyme (144 mg) now had a specific activity of 46 units/mg of protein. It was adsorbed onto a Mono Q HR column and chromatographed with a 0–1 m KCl gradient. A peak of activity (8 mg, specific activity = 186 units/mg of protein) appeared around 0.2m KCl. After concentration in Centricon 30 tubes, the protein was adsorbed onto a 10-ml column of hydroxylapatite equilibrated with 50 mm Tris-HCl, pH 8.0. Inactive protein was removed by elution with 75 mm phosphate, pH 7.5, followed by elution of the active protein (1.7 mg, specific activity = 246 units/mg of protein) with 200 mmphosphate, pH 7.5. After concentration in Centricon 30 tubes, the protein was equilibrated with 0.1 m KCl, 50 mmTris-HCl, pH 7.5, and 10% glycerol on a 5-ml column of Sephadex G-25, giving a final yield of 0.51 mg of pure enzyme. The protein was highly “sticky,” and recovery was low. It was also prepared in pure form, but again in low yield, by affinity chromatography on dATP-Sepharose as a final purification step instead of hydroxylapatite in a procedure related to the one described below for the T. maritimareductase. Both preparations gave a single band on denaturing gels and, when used in binding experiments, gave identical results. After ammonium sulfate precipitation, the protein in buffer B (1.44 g, 10 mg/ml, specific activity = 70 units/mg of protein) was heated for 10 min at 80 °C, and the resulting precipitate was removed by high speed centrifugation. After addition of 1.15 ml of 1 mCaCl2, the solution (114 ml, 1 mg of protein/ml, specific activity = 550 units/mg of protein) was adsorbed onto a 25-ml column of dATP-Sepharose equilibrated with 0.1 m KCl, 30 mm Tris-HCl, pH 7.5, 10 mm CaCl2, and 2 mm DTT. The column was washed with 40 ml of the same buffer, and the reductase was eluted with 0.5 m ammonia. The combined fractions containing 29 mg of protein were neutralized with 2 m NaH2PO4, and the protein was precipitated overnight after addition of solid ammonium sulfate to 70% saturation. Denaturing gel electrophoresis at this stage showed only two protein bands with mobilities of ∼70 and 90 kDa. The proteins corresponding to these bands could be separated by chromatography on a column of Superdex 200 (Amersham Pharmacia Biotech) equilibrated with 0.1 m KCl, 30 mm Tris-HCl, pH 7.5, and 2 mm DTT. The ammonium sulfate precipitate was dissolved in 1.5 ml of this buffer and added to the column. Elution with the same buffer resulted in the appearance of two cleanly separated symmetrical protein peaks with mobilities corresponding to ∼500 and 150 kDa, named TM1 and TM2, both enzymatically active. The materials corresponding to each peak were concentrated in Centriprep 30 tubes. TM1 (8.5 mg, specific activity = 1500 units/mg of protein) corresponded to the 90-kDa band on denaturing gels. Mass spectrometry showed a single peak with a mass of 93.5 kDa. 2A. Jordan, R. Eliasson, U. Hellman, I. Gibert, and P. Reichard, unpublished results. TM2 (2.9 mg, specific activity = 1800 units/mg of protein) corresponded to the 70-kDa band. Both TM1 and TM2 had the same N-terminal sequence as the enzyme isolated from T. maritima,2 suggesting that the smaller size of TM2 was due to C-terminal processing of the enzyme. Under standard conditions, all three enzymes were incubated in a final volume of 50 μl for 20 min in the presence of 0.5 mm [3H]CTP (L. leichmanniireductase) or [3H]CDP (T. acidophilum and T. maritima reductases), 100 mm DTT, 15 μm adenosylcobalamin, 30 mm Tris-HCl, pH 8.0, 100 μm dATP, and 10 mm CaCl2(L. leichmannii enzyme), 10 mm MgCl2(T. maritima enzyme), or 40 mm MgCl2(T. acidophilum enzyme) at 37 °C (L. leichmannii enzyme), 80 °C (T. maritima enzyme), or 55 °C (T. acidophilum enzyme). The reaction was terminated with 1 ml of ice-cold 1 m HClO4, and the amount of [3H]dCMP formed was determined by chromatography on Dowex-50 after 10 min of hydrolysis at 100 °C (22Thelander L. Sjöberg B.-M. Eriksson S. Methods Enzymol. 1978; 51: 227-237Crossref PubMed Scopus (71) Google Scholar). Reduction of ADP (ATP) or GDP (GTP) was assayed with14C-labeled substrates under identical incubation conditions except for the allosteric effector. After incubation, the nucleotides were transformed to nucleosides by digestion with alkaline phosphatase, and the labeled deoxyribosides were separated from the ribosides on boronate columns (23Kim J.J. Abrams R. Franzen J.S. Arch. Biochem. Biophys. 1977; 182: 674-682Crossref PubMed Scopus (5) Google Scholar). One unit of activity is defined as 1 nmol of product formed during 1 min. Specific activity is units/mg of protein. Protein was determined colorimetrically (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with crystalline bovine serum albumin as the standard. 5–20% linear sucrose gradients in a total volume of 4.6 ml were prepared in 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, and 2 mm DTT. In some experiments, 15 μm adenosylcobalamin and/or 100 μm dATP was present throughout the gradient. Solutions (0.2 ml) of the enzymes containing catalase as an internal marker (s20,w = 11.4 S) were layered onto the gradient and centrifuged in a Beckman SW 50 rotor at 32,000 rpm for 18 h at 20 °C. After the run, the tubes were punctured at the bottom, and a total of 33–38 fractions were collected and analyzed for protein content and catalase activity. The sedimentation coefficients of the proteins were calculated from their positions in the gradient relative to the catalase marker. The method of Ormö and Sjöberg (25Ormö M. Sjöberg B.-M. Anal. Biochem. 1990; 182: 674-682Google Scholar) was used at +4 °C as described previously (17Eliasson R. Pontis E. Jordan A. Reichard P. J. Biol. Chem. 1996; 271: 26582-26587Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Eliasson R. Pontis E. Sun X. Reichard P. J. Biol. Chem. 1994; 269: 26052-26057Abstract Full Text PDF PubMed Google Scholar). No dephosphorylation of any nucleotide occurred during the course of the experiments as determined by polyethyleneimine chromatography. Enzyme purification as described under “Experimental Procedures” provided large amounts of L. leichmannii and T. maritimareductases, but only limited amounts of the T. acidophilumenzyme. All three enzymes gave rise to a single band on denaturing gel electrophoresis (Fig. 1) with positions in accordance with their molecular masses of 82, 94, and 97 kDa for the enzymes from L. leichmannii (10Booker S. Stubbe J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8352-8356Crossref PubMed Scopus (96) Google Scholar), T. maritima(14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar), and T. acidophilum (21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 53-58Crossref PubMed Scopus (50) Google Scholar), respectively, calculated from their amino acid compositions. In the case of the T. maritima reductase, we separated two homogeneous highly active enzymes with different molecular masses: one with a mass of 94 kDa corresponding to the enzyme prepared from T. maritima and a second shorter protein of ∼70 kDa that had apparently arisen through C-terminal processing of the larger protein in E. coli. The large protein was used in our experiments; the properties of the shorter one will be described elsewhere.2The specific activities of the pure L. leichmannii and T. acidophilum enzymes differed from earlier reported values. The L. leichmannii reductase showed only half the activity; the T. acidophilum reductase was 10 times more active. We ascribe this to differences in assay conditions. The reductant in our assays was DTT, whereas thioredoxin from E. coli was employed earlier with the L. leichmanniireductase. In the case of the T. acidophilum reductase, we optimized both the DTT and cation concentrations better than was done in previous work. Extensive experiments from two separate laboratories (9Panagon D. Orr M.D. Dunstone J.R. Blakeley R.L. Biochemistry. 1972; 11: 2378-2388Crossref PubMed Scopus (57) Google Scholar, 20Chen A.K. Bhan A. Hopper S. Abrams R. Franzen J.S. Biochemistry. 1974; 13: 654-661Crossref PubMed Scopus (34) Google Scholar) indicated that the native L. leichmannii enzyme is a monomer with a molecular mass close to 80 kDa. Also the native T. acidophilum enzyme was briefly stated to be a monomer (21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 53-58Crossref PubMed Scopus (50) Google Scholar), whereas the T. maritima enzyme behaved as a high molecular mass oligomer (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar). As the oligomeric state of the reductases is important for an understanding of the allosteric effects, we decided to re-determine this parameter. To this purpose, we measured the sedimentation of the enzymes in sucrose gradients under various conditions that might affect their aggregation, including different combinations of adenosylcobalamin, dATP, DTT, and Mg2+. We had used sucrose gradient centrifugation with advantage earlier to determine the oligomeric structures of class I (26Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 25-38Crossref PubMed Scopus (93) Google Scholar) and III (13Ollagnier S. Mulliez E. Gaillard J. Eliasson R. Fontecave M. Reichard P. J. Biol. Chem. 1996; 271: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) ribonucleotide reductases. Gradients were standardized with the R1E protein of Salmonella typhimurium (161 kDa,s20,w = 7.7–7.9 S), aldolase (158 kDa, 7.9 S), and bovine serum albumin (67 kDa, 4.8 S). Irrespective of conditions, the L. leichmannii enzyme (monomer = 82 kDa) sedimented at 5.6–5.7 S, the T. acidophilum enzyme (monomer = 97 kDa) at 8.4–8.6 S, and the T. maritimaenzyme (monomer = 94 kDa) at 10.2 S. All runs were made at room temperature, but the T. maritima enzyme was heated to 80 °C for 10 min in the presence of 20 μmadenosylcobalamin, 2 mm DTT, 0.1 mm dATP, and 10 mm MgCl2 before centrifugation. The sedimentation coefficients of the three reductases suggest that, in solution, the L. leichmannii enzyme is a monomer, the T. acidophilum enzyme a dimer, and the T. maritima enzyme a higher oligomer. As the T. maritimaenzyme normally operates above 80 °C and centrifugations were at room temperature, more extensive studies are required to establish its true oligomeric state. Our aim was to study the influence of allosteric modulators on the substrate specificity and activity of the reductases under optimal assay conditions. We first optimized the reduction of CDP (CTP) with each enzyme and then used the results to study effector requirements for the reduction of purine ribonucleotides. As reported earlier (9Panagon D. Orr M.D. Dunstone J.R. Blakeley R.L. Biochemistry. 1972; 11: 2378-2388Crossref PubMed Scopus (57) Google Scholar, 14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar, 21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 53-58Crossref PubMed Scopus (50) Google Scholar), the activity of each of the three enzymes is completely dependent on the presence of adenosylcobalamin and high concentrations (100 mm) of DTT. Substrates for the reaction are ribonucleosidediphosphates for the T. acidophilum and T. maritima reductases (14Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar, 21Tauer A. Benner S.A. Proc. Natl. Acad. Sci. U" @default.
• W2037487893 created "2016-06-24" @default.
• W2037487893 creator A5003486360 @default.
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• W2037487893 date "1999-03-01" @default.
• W2037487893 modified "2023-09-30" @default.
• W2037487893 title "Allosteric Control of Three B12-dependent (Class II) Ribonucleotide Reductases" @default.
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