The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m312018200
ISSN1083-351X
AutoresTakayoshi Iwai, Seiki Kuramitsu, Ryoji Masui,
Tópico(s)Carbon and Quantum Dots Applications
ResumoThe ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 °C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap6A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap6A, with a Km of 1.4 μm and a kcat of 4.1 s–1. These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the divalent cations Mn2+, Mg2+, Zn2+, and Co2+, whereas Ca2+, Ni2+, and Cu2+ were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for Ap6A was observed at around pH 8.0 and about 70 °C. We found two important residues with pKa values of 6.1 and 9.6 in the free enzyme and pKa values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed. The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 °C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap6A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap6A, with a Km of 1.4 μm and a kcat of 4.1 s–1. These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the divalent cations Mn2+, Mg2+, Zn2+, and Co2+, whereas Ca2+, Ni2+, and Cu2+ were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for Ap6A was observed at around pH 8.0 and about 70 °C. We found two important residues with pKa values of 6.1 and 9.6 in the free enzyme and pKa values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed. The Nudix hydrolase family comprises enzymes that catalyze a reaction where the substrate is a nucleoside diphosphate linked to another moiety, X (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). This family is characterized by the Nudix motif GX5EX7REUXEEXGU, where X is any amino acid, and U is one of the bulky hydrophobic amino acids, Ile, Leu, or Val (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). This motif forms a loop-helix-loop structure that is involved in substrate binding and catalysis (2Lin J. Abeygunawardana C. Frick D.N. Bessman M.J. Mildvan A.S. Biochemistry. 1997; 36: 1199-1211Crossref PubMed Scopus (82) Google Scholar, 3Gabelli S.B. Bianchet M.A. Bessman M.J. Amzel L.M. Nat. Struct. Biol. 2001; 8 (L. M.): 467-472Crossref PubMed Scopus (113) Google Scholar). These enzymes are found in all kingdoms. It has been proposed that the function of Nudix proteins is housecleaning to eliminate potentially toxic nucleotide metabolites from the cell and to regulate the concentrations of nucleoside diphosphate derivatives (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). A recent Pfam (4Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2015) Google Scholar) search of the data bases for the Nudix signature sequence has revealed about 1100 open reading frames from more than 250 species ranging from viruses to humans, and about 70 of the gene products have been identified. These products hydrolyze nucleoside diphosphate derivatives such as (deoxy)nucleoside triphosphate (5O'Handley S.F. Frick D.N. Bullions L.C. Mildvan A.S. Bessman M.J. J. Biol. Chem. 1996; 271: 24649-24654Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), nucleotide sugar (6O'Handley S.F. Frick D.N. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 3192-3197Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Frick D.N. Townsend B.D. Bessman M.J. J. Biol. Chem. 1995; 270: 24086-24091Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Yang H. Slupska M.M. Wei Y.F. Tai J.H. Luther W.M. Xia Y.R. Shih D.M. Chiang J.H. Baikalov C. Fitz-Gibbon S. Phan I.T. Conrad A. Miller J.H. J. Biol. Chem. 2000; 275: 8844-8853Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), dinucleotide polyphosphate (9Maksel D. Guranowski A. Ilgoutz S.C. Moir A. Blackburn M.G. Gayler K.R. Biochem. J. 1998; 329: 313-319Crossref PubMed Scopus (37) Google Scholar, 10Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Crossref PubMed Scopus (34) Google Scholar, 11Thorne N.M. Hankin S. Wilkinson M.C. Nunez C. Barraclough R. McLennan A.G. Biochem. J. 1995; 311: 717-721Crossref PubMed Scopus (59) Google Scholar), NADH (12Frick D.N. Bessman M.J. J. Biol. Chem. 1995; 270: 1529-1534Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and coenzyme A (13AbdelRaheim S.R. McLennan A.G. BMC Biochem. 2002; 3: 5Crossref PubMed Scopus (20) Google Scholar). One example is the MutT protein, which degrades 8-oxo-deoxyguanine triphosphate to prevent mutations caused by oxidation of guanine nucleotides (14Maki H. Sekiguchi M. Nature. 1992; 355: 273-275Crossref PubMed Scopus (790) Google Scholar). Furthermore, diphosphoinositol polyphosphate (DIPP) 1The abbreviations used are: DIPP, diphosphoinositol polyphosphate; Ap6A, diadenosine hexaphosphate; Ap5A, diadenosine pentaphosphate; Ap4A, diadenosine tetraphosphate; Ap3A, diadenosine triphosphate; Gp5G, diguanosine pentaphosphate; Gp4 G, diguanosine tetraphosphate; p4A, adenosine tetraphosphate; CD, circular dichroism; ApnA (n is the number of phosphate groups), diadenosine polyphosphate; h-, human. (15Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar) and phosphoribosyl pyrophosphate have also been reported as substrates for Nudix hydrolases (16Fisher D.L. Safrany S.T. McLennan A.G. Cartwright J.L. J. Biol. Chem. 2002; 277: 47313-47317Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Although preferred substrates have been identified for some Nudix enzymes, the functions and molecular mechanisms, including substrate recognition, remain to be elucidated. Thermus thermophilus HB8 is a Gram-negative bacterium that grows at temperatures above 75 °C (17Oshima T. Imahori K. Int. J. Syst. Bacteriol. 1974; 24: 102-112Crossref Scopus (492) Google Scholar). It is the most thermophilic eubacterium for which a gene manipulation system has been established (18Yamagishi A. Tanimoto T. Suzuki T. Oshima T. Appl. Environ. Microbiol. 1996; 62: 2191-2194Crossref PubMed Google Scholar, 19Hoseki J. Yano T. Koyama Y. Kuramitsu S. Kagamiyama H. J. Biochem. (Tokyo). 1999; 126: 951-956Crossref PubMed Scopus (145) Google Scholar, 20Hashimoto Y. Yano T. Kuramitsu S. Kagamiyama H. FEBS Lett. 2001; 506: 231-234Crossref PubMed Scopus (86) Google Scholar). Proteins from this bacterium are stable against heat and are, thus, suitable for physicochemical studies, including x-ray crystallography. We selected T. thermophilus HB8 for the systematic study of the structures and functions of all proteins from a single organism in a project named the Whole Cell Project (21Yokoyama S. Hirota H. Kigawa T. Yabuki T. Shirouzu M. Terada T. Ito Y. Matsuo Y. Kuroda Y. Nishimura Y. Kyogoku Y. Miki K. Masui R. Kuramitsu S. Nat. Struct. Biol. 2000; 7: 943-945Crossref PubMed Scopus (325) Google Scholar, 22Yokoyama S. Matsuo Y. Hirota H. Kigawa T. Shirouzu M. Kuroda Y. Kurumizaka H. Kawaguchi S. Ito Y. Shibata T. Kainosho M. Nishimura Y. Inoue Y. Kuramitsu S. Prog. Biophys. Mol. Biol. 2000; 73: 363-376Crossref PubMed Scopus (47) Google Scholar). Interestingly, Deinococcus radiodurans, which is very closely related to T. thermophilus, possesses twenty-three Nudix genes (23White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Minton K.W. Fleischmann R.D. Ketchum K.A. Nelson K.E. Salzberg S. Smith H.O. Craig Venter J. Fraser C.M. Science. 1999; 286: 1571-1577Crossref PubMed Scopus (797) Google Scholar). This bacterium is characterized by extraordinary resistance to ionizing radiation; it is thought that some of its Nudix proteins may be associated with novel DNA repair pathways (24Xu W. Shen J. Dunn C.A. Desai S. Bessman M.J. Mol. Microbiol. 2001; 39: 286-290Crossref PubMed Scopus (58) Google Scholar). However, the diversity of substrates and functions of Nudix proteins in vivo remain unclear. Such unique features make proteins of this family good targets for structural and functional proteomics. Therefore, we aimed to investigate the molecular mechanism and physiological functions of Nudix proteins from T. thermophilus HB8. In this work, we describe the overexpression and purification of T. thermophilus HB8 Ndx1 protein. The enzymatic activity and the biochemical properties of Ndx1 are also described. Materials—DNA-modifying enzymes, including restriction enzymes, were from Takara Shuzo and New England Biolabs. Yeast extract and polypeptone were from Difco. Isopropyl-β-d-thiogalactopyranoside was from Wako Pure Chemicals. Escherichia coli strains BL21(DE3) and DH5α and plasmids pET-11b and pT7Blue were from Novagen. Toyopearl-SuperQ and Toyopearl-Phenyl 650M were from Tosoh. Superdex 75 10/30 was from Amersham Bioscience. The CAPCELL PAK C18 column was from Shiseido. The molecular weight marker kit was from Sigma. Dinucleoside polyphosphates (diadenosine hexaphosphate (Ap6A), diadenosine pentaphosphate (Ap5A), diadenosine tetraphosphate (Ap4A), diadenosine triphosphate (Ap3A), diguanosine pentaphosphate (Gp5G), diguanosine tetraphosphate (Gp4G)) and other nucleotide derivatives (adenosine tetraphosphate (p4A), ATP, GTP, ADP-ribose, NADH, NAD+, CoA, and acetyl CoA) were from Sigma. TaKaRa LA Taq was from Takara Shuzo. The synthesized DNA oligomers were from BEX Co. All other reagents used were of the highest grade commercially available. Overexpression of the ndx1 Gene—Preliminary sequence data for the T. thermophilus HB8 ndx1 gene, which contains the Nudix motif, was provided by the T. thermophilus HB8 genome project (21Yokoyama S. Hirota H. Kigawa T. Yabuki T. Shirouzu M. Terada T. Ito Y. Matsuo Y. Kuroda Y. Nishimura Y. Kyogoku Y. Miki K. Masui R. Kuramitsu S. Nat. Struct. Biol. 2000; 7: 943-945Crossref PubMed Scopus (325) Google Scholar, 22Yokoyama S. Matsuo Y. Hirota H. Kigawa T. Shirouzu M. Kuroda Y. Kurumizaka H. Kawaguchi S. Ito Y. Shibata T. Kainosho M. Nishimura Y. Inoue Y. Kuramitsu S. Prog. Biophys. Mol. Biol. 2000; 73: 363-376Crossref PubMed Scopus (47) Google Scholar). Using this information, two primers for amplification of the target gene were synthesized, and PCR (polymerase chain reaction) was carried out using these primer and LA Taq polymerase. The primer sequences were 5′-ATATCATATGGAGCTAGGGGCCGGGGGCGTGGTCTT-3′ and 5′-ATATAGATCTTTATTAAAGCGGTAGACGCTCAAGGG-3′, and the underlining indicates NdeI and BglII sites. The amplified gene fragment was ligated into pET-11b (Novagen) using NdeI and BamHI sites following TA cloning and sequencing. E. coli BL21(DE3) cells transformed by the resulting plasmid were grown at 37 °C to 5 × 108 cell/ml on 1.5 liters of LB medium containing ampicillin. The cells were then incubated for 4 h in the presence of isopropyl-β-d-thiogalactopyranoside, harvested by centrifugation, and stored at –20 °C. Purification of Ndx1—All purification steps described below were carried out at room temperature. Frozen cells (3 g) were suspended in 30 ml of lysis buffer (50 mm Tris-HCl, 0.1 mm EDTA, 10% (w/v) glycerol, and 20% (w/v) saccharose (pH 8.5)) and disrupted by ultrasonication on ice. After Brij-58 was added to a final concentration of 0.2% (w/v), the cell lysate was stirred for 1 h at 4 °C. Next, the lysate was incubated at 70 °C for 10 min and centrifuged (about 30,000 × g) for 60 min. Then the supernatant was dialyzed against buffer I (50 mm Tris-HCl, 0.1 mm EDTA, and 10% (w/v) glycerol (pH 8.5)) for 16 h. The dialysate was applied to a Toyopearl-SuperQ column (bed volume 12 ml) that had been equilibrated with buffer I. Proteins were eluted with a linear gradient of NaCl from 0 to 1.0 m in a total volume of 60 ml of buffer I. Solid ammonium sulfate was added to the fractions containing the Ndx1 protein to a final concentration of 15% saturation. The protein solution was then applied to a Toyopearl-Phenyl 650M column (bed volume 10 ml) previously equilibrated with buffer I containing 15% saturated ammonium sulfate. The proteins were eluted with a linear gradient of ammonium sulfate from 15 to 0% saturation in a total volume of 50 ml of buffer I. Fractions containing the Ndx1 protein were collected and concentrated using a Vivaspin (5000 cut off) concentrator. The concentrated solution was applied to a Superdex 75 10/30 column (bed volume 24 ml) previously equilibrated with buffer II (50 mm Tris-HCl and 100 mm KCl (pH 7.5)) and eluted with the same buffer using ΔKTA explorer (Amersham Biosciences). The fraction containing the Ndx1 protein was concentrated and stored at 4 °C. At each chromatography, the elution profile was assessed by SDS-PAGE containing 12% (w/v) acrylamide. The N-terminal sequence of the purified Ndx1 was analyzed on an Applied Biosystems 473A protein sequencer. CD Spectrometry—Circular dichroism (CD) measurements were carried out with a Jasco spectropolarimeter, model J-720W. CD spectra of 5 μm Ndx1 were measured in a 1-mm cell in the far-UV region between 200 and 250 nm. Measurements were performed after incubation at 25 °C in 50 mm potassium phosphate (pH 7.5) and 100 mm KCl. Thermostability was assessed by measuring CD values at 222 nm at a 1 °C/min rate. CD data were converted to the mean residue ellipticity, [θ], in deg cm2 dmol–1. Size Exclusion Chromatography—The oligomeric state of Ndx1 in solution was assessed by size exclusion chromatography on Superdex 75 HR 10/30. A sample contained 50 mm Tris-HCl (pH 7.5), 100 mm KCl, and 0.1 μm Ndx1, which were the same conditions used to assay Ndx1 activity. The protein was eluted with 50 mm Tris-HCl and 100 mm KCl (pH 7.5) with a flow rate of 0.5 ml/min by the ΔKTA system. The molecular weight of Ndx1 was estimated using molecular weight marker proteins (Sigma). Hydrolase Activity Assay—The activity of Ndx1 was measured by quantifying the amounts of substrates and products with an ion-pair reversed-phase high performance chromatography according to a slightly modified method of Samizo et al. (25Samizo K. Ishikawa R. Nakamura A. Kohama K. Anal. Biochem. 2001; 293: 212-215Crossref PubMed Scopus (31) Google Scholar). Reaction mixtures (100 μl) containing 50 mm Tris-HCl, 100 mm KCl, 5 mm MgCl2, substrate, and Ndx1 were incubated at 25 °C. The reaction was stopped by adding 100 μl of 100 mm EDTA, and the protein was removed by ultrafiltration using a membrane filter. The 100-μl aliquot of the filtrate was applied to a reversed-phase column (CAPCELL PAK C18, 4.6 × 75 mm), which was equilibrated with 20 mm sodium phosphate (pH 7.0), 5 mm tetra-n-butyl ammonium phosphate, and 10% methanol. Elution was performed by a gradient of 10–50% methanol. Nucleotides were detected at 260 nm, and their identifications were based on their retention times. Their concentrations were calculated by the integration of their respective peak areas. Concentrations of substrates were varied between 0.2 and 16 μm. Initial velocity was calculated from product concentration and plotted against substrate concentration. These were fitted to the Michaelis-Menten equation and Hanes-Woolf plot, and the kinetic constant was calculated using the software Igor Pro 3.14 (Wave Metrics). Fitting Plot of pH-dependent Assay—Assuming Scheme 1 for the Ndx1 reaction, the following equations could be generated (26Fersht A.R. Structure and Mechanism in Protein Science. W. H. Freeman and Co., New York1999: 169-190Google Scholar). In short, there are two residues that are related to catalysis by Ndx1. Km=Ks(1+[H+]/Kel+Ke2/[H+])/(1+[H+]/Kes1+Kes2/[H+])(Eq. 1) V=k2[E0]/(1+[H+]/Kes1+Kes2/[H+])(Eq. 2) kcat=k2/(1+[H+]/Kes1+Kes2/[H+])(Eq. 3) V/Km=k2[E0]/{Ks(1+[H+]/Ke1+Ke2/[H+])}(Eq. 4) kcat/Km=k2/{Ks(1+[H+]/Ke1+Ke2/[H+])}(Eq. 5) The pKa values of one residue are pKe1 and pKes1 at free enzyme and complex, respectively. Similarly, those of the other residue are pKe2 and pKes2. These pKa values were calculated by fitting the data to Equations 1, 2, 3 using the software Igor Pro 3.14 (Wave Metrics) Fluorescence Measurements—The fluorescence emission of Ndx1 was measured with a Hitachi spectrofluorometer, model F-4500. All measurements were taken with an excitation wavelength of 295 nm in a 5 × 5-mm quartz cuvette at 25 °C. The fluorescence titration was carried out by measuring the emission intensity at 328 nm. The reaction mixture (200 μm) contained 50 mm Tris-HCl, 100 mm KCl, 5 mm MgCl2, 0.5 μm Ndx1, and various concentrations of ATP (pH 7.5). Site-directed Mutagenesis—Pairs of oligonucleotides about 30 bases in length with melting temperatures of about 67 °C and containing the desired substitutions were designed. The ndx1 gene includes NruI and PshAI sites at the 60th and 155th positions, respectively. For W26A substitution, the region between the NruI site and BglII site at the 3′ terminus of the gene was amplified by PCR. For R45K, E46Q, E49Q, and E50Q substitution, the regions between the 5′ terminal NdeI site and PshAI site were amplified. The amplified DNA fragments were cloned into the pT7Blue vector by TA cloning. E. coli DH5α were transformed with the constructed plasmid DNA and cultured. Substitution at the desired positions was confirmed by sequencing. Then the wild-type fragments in the expression vector were replaced by the confirmed fragments containing the mutations. Preparation of Ndx1—Using information from the T. thermophilus HB8 genome project, we identified eight open reading frames containing Nudix motifs and named them ndx1 to ndx8. The ndx1 gene product (DDBJ/EMBL/GenBank™ accession number AB125632; project code 1331) comprises 126 amino acids, has a molecular mass of 14.2 kDa, and has a theoretical pI of 4.8. When a BLAST search was carried out using the Ndx1 sequence as a query, Ndx1 was the most similar (about 25% identity) to Ap4A hydrolases from Caenorhabditis elegans, human, and pig (Fig. 1A). However, the sequence similarity was restricted to the surroundings of the Nudix motif and was not enough to determine whether Ndx1 is an Ap4A hydrolase or not. Thus, Ndx1 was overexpressed in E. coli BL21(DE3) under the control of an isopropyl-β-d-thiogalactopyranoside -inducible T7 promoter. The induced band at ∼15 kDa (corresponding to the size of Ndx1) was observed in the soluble fraction (Fig. 2), and we purified the protein to homogeneity utilizing three column chromatography steps (see details under "Experimental Procedures"). The sequence of the N-terminal nine residues of the overexpressed protein agreed with the residues predicted from the ndx1 sequence, confirming that the purified protein was Ndx1. Approximately 25 mg of Ndx1 was obtained from 3 g of cells. Physicochemical Properties—Size exclusion chromatography was performed to investigate the oligomeric state of Ndx1. The elution profile of Ndx1 showed a single peak (Fig. 2B). The apparent molecular mass corresponding to the peak was estimated to be 17 kDa, which was similar to the 14.2-kDa mass calculated from the sequence. This indicates that Ndx1 exists in a monomeric state in solution. The far-UV CD spectrum of the purified Ndx1 showed negative double maxima at 209 and 220 nm (Fig. 3A), characteristic of an α-helical structure. The stability of Ndx1 to temperature and pH was examined based on the mean residue ellipticity at 222 nm ([θ]222). Ndx1 was stable up to 95 °C at pH 7.5 (Fig. 3B) and stable in a wide range of pH at 25 °C (Fig. 3C). Enzymatic Activity—Most Nudix proteins examined to date are nucleotide pyrophosphatases that hydrolyze a nucleoside diphosphate linked to another moiety (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Therefore, enzymatic activity of Ndx1 was examined for a wide range of nucleotides known to be substrates of other Nudix proteins. Ndx1 was inactive toward the following nucleotides when assayed at 50 μm: 5′-(deoxy)nucleoside triphosphates, 5′-nucleoside diphosphates, nucleoside diphosphate sugars, NADH, NAD+, CoA, and acetyl CoA. Significant activity was found toward dinucleotide polyphosphates and nucleotide polyphosphates. The respective substrates yielded products as follows: Ap6A, 2ATP; Ap5A, ATP and ADP; Ap4A, ATP and AMP; p4A, ATP and inorganic orthophosphate (Table I). In all cases ATP was generated as a product. ATP and Ap3A were not hydrolyzed by Ndx1. These data indicate that Ndx1 protein has ATP-generating (di)nucleotide polyphosphate hydrolase activity.Table IKinetic constants for Ndx1SubstrateProductkcatKmkcat/ Kms-1μmm-1 s-1Ap6AATP4.11.42.9 × 106p4AATP, Pi1.41.01.4 × 106Ap5AATP, ADP0.521.14.7 × 105Ap4AATP, AMP0.271.12.5 × 105Gp5GGTP, GDP0.271.42.0 × 105Gp4GGTP, GMP0.099.39.7 × 103 Open table in a new tab Table I shows the steady-state kinetic constants for each active substrate assuming Michaelis-Menten type reactions. Whereas the Km values for these substrates were all about 1 μm, the catalytic constants (kcat) varied among the tested substrates (Table I). Based on the catalytic efficiencies (kcat/Km), the highest activity was observed for Ap6A followed by p4A, Ap5A, and Ap4A (Table I). The substrate preference was Ap6A > p4A ≫ Ap5A > Ap4A for polyphosphates and adenosine > guanosine for the base. Therefore, we conclude that Ndx1 is an ATP-generating Ap6A hydrolase. Among the known enzymes that specifically hydrolyze Ap6A, Ndx1 is the only enzyme that symmetrically hydrolyzes Ap6A. Divalent metal ions were essential for Ndx1 activity, which is a common property of Nudix proteins (27Frick D.N. Weber D.J. Gillespie J.R. Bessman M.J. Mildvan A.S. J. Biol. Chem. 1994; 269: 1794-1803Abstract Full Text PDF PubMed Google Scholar). The effect of several divalent metal ions (each 5 mm) on Ndx1 activity for Ap6A was investigated. Significant activity was observed in the presence of Mn2+, Mg2+, and Zn2+ with the apparent rate constant of kapp values of 8.6, 4.2, and 4.1 s–1, respectively. In contrast, there was low activity in the presence of Co2+ (kapp, 0.51 s–1), and there was no activity in the presence of Ca2+, Ni2+, and Cu2+. Among monovalent ions, fluoride ion showed strong inhibition of the Ndx1 activity. Dependence of the inhibitory effect on fluoride ion concentration revealed that the inhibition was in a non-competitive manner with a Ki of 424 μm toward free enzyme and a Ki of 80 μm toward complex. Ndx1 exhibited higher activity at higher pH and little activity at lower pH. This observation is typical for the Nudix class of enzymes, which usually have optimal pH values in the alkaline range. The presence of two pKa values (7.9 and 10.0) was found in the plot of kcat against pH (Fig. 4A). The optimal pH for Ndx1 activity was about 8, judged by the catalytic efficiency kcat/Km (Fig. 4B). The plot of the kapp against temperature was bell-shaped; the optimal temperature for Ndx1 activity was 70 °C. This higher activity at higher temperature reflects a common property of enzymes from T. thermophilus. Protein-Substrate Interaction—The Km values of Ndx1 for several (di)adenosine polyphosphates were about 1 μm. Because these results suggest that the affinity for these substrates is almost the same, we hypothesized that Ndx1 recognized a common moiety of the substrates at the initial binding phase. Although several tertiary structures of Nudix proteins have been reported (2Lin J. Abeygunawardana C. Frick D.N. Bessman M.J. Mildvan A.S. Biochemistry. 1997; 36: 1199-1211Crossref PubMed Scopus (82) Google Scholar, 3Gabelli S.B. Bianchet M.A. Bessman M.J. Amzel L.M. Nat. Struct. Biol. 2001; 8 (L. M.): 467-472Crossref PubMed Scopus (113) Google Scholar, 28Fletcher J.I. Swarbrick J.D. Maksel D. Gayler K.R. Gooley P.R. Structure. 2002; 10: 205-213Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Swarbrick J.D. Bashtannyk T. Maksel D. Zhang X.R. Blackburn G.M. Gayler K.R. Gooley P.R. J. Mol. Biol. 2000; 302: 1165-1177Crossref PubMed Scopus (47) Google Scholar), detailed investigations of substrate binding mechanisms have not often been performed by biochemical methods. The adenosine phosphate moiety is commonly contained in Ndx1 substrates, and Ndx1 has no activity toward ATP. Therefore, we measured the intrinsic fluorescence spectrum of Ndx1 upon binding to ATP to investigate the interaction between the protein and the adenosine phosphate moiety. The emission spectra were measured using an excitation wavelength of 295 nm, which excited only tryptophan residues. When ATP was added to Ndx1, the fluorescence intensity at around 328 nm decreased (Fig. 5A, bold solid line). As the ATP concentration increased, the fluorescence intensity gradually decreased. These results suggest that the decrease in fluorescence intensity reflects the protein-substrate interaction. When dATP was used as a ligand, the fluorescence intensity also decreased but not equivalently to ATP. When the change in fluorescence intensity was plotted against ligand concentration, Kd was determined to be 13 μm for ATP and 36 μm for dATP based on bimolecular binding reaction (Fig. 5B). The affinity of Ndx1 for these nucleotides was also investigated by examining the inhibition of ATP and dATP on Ap6A hydrolysis by Ndx1 (not data shown). These results showed that Ki was 13 μm for ATP and 41 μm for dATP. When Mg2+ was omitted from the reaction mixture, no fluorescence change was observed (Fig. 5A, dotted line). Also, when GTP was used, the fluorescence intensity did not decrease, as is predicted by the low affinity of Ndx1 toward diguanosine polyphosphates. Among the four Trp residues of Ndx1, Trp-26 was conserved in the N-terminal half of the Nudix motif in the sequence of dinucleotide polyphosphate hydrolases (Fig. 1B). This raised the possibility that the observed fluorescence changes could be ascribed to Trp-26. This hypothesis was confirmed by the observation that the mutant W26A Ndx1, in which Trp-26 was replaced by alanine, showed no decrease in fluorescence intensity upon the addition of ATP (Fig. 5C). Catalytic Residues—From the dependence of Ndx1 hydrolysis activity on pH, pKa values of 7.9 and 10.0 were obtained from the kcat plot (Fig. 4A), and pKa values of 6.1 and 9.6 were obtained from the plot of kcat/Km (Fig. 4B). These pKa changes demonstrated that when free Ndx1 bound to the substrate, the pKa of one residue changed from 6.1 to 7.9, and the pKa of a second residue changed from 9.6 to 10.0. It is thought that two residues whose pKa values change play an important role in Ndx1 hydrolysis. In Ndx1, the conserved Nudix motif is located between Gly-31 and Val-53. In this motif, Glu-37, Arg-45, Glu-46, Glu-49, and Glu-50 are highly conserved across the sequences of Nudix proteins from plants, animals, and bacteria (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). When the glutamic acid residues (Glu-46, Glu-49, and Glu-50) in the Nudix motif were replaced by glutamine, the E46Q and E50Q mutants showed a 2.2 × 104-fold reduction and a 1.3 × 105-fold reduction in kcat, respectively (Table II). In contrast, the E49Q mutation had very little effect on activity (Table II). Moreover, mutation of the conserved Arg-45 residue to lysine only slightly reduced the activity (Table II).Table IIKinetic constants for Ap6A hydrolysis by mutant Ndx1MutantkcatKmkcat/ Kms-1μmm-1 s-1Wild type4.11.42.9 × 106W26A1.5197.8 × 104R45K5.5 × 10-1115.0 × 104E46Q8.9 × 10-41.65.6 × 102E49Q1.11.66.8 × 105E50Q5.2 × 10-52
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