Studies on the ADP-ribose Pyrophosphatase Subfamily of the Nudix Hydrolases and Tentative Identification of trgB, a Gene Associated with Tellurite Resistance

Four Nudix hydrolase genes, ysa1 fromSaccharomyces cerevisiae, orf209 fromEscherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite. Four Nudix hydrolase genes, ysa1 fromSaccharomyces cerevisiae, orf209 fromEscherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite. The Nudix hydrolases comprise a large family of proteins characterized by the highly conserved array of amino acids GX5EX7REUXEEXGU, where U represents a bulky, hydrophobic, amino acid, usually 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 (583) Google Scholar). A recent BLAST (2Altschul S.F. Gish W. Meyers E.W. Lipman D.J. J. Mol. Biol. 1990; 203: 403-410Crossref Scopus (69694) Google Scholar) search of the sequence data banks has revealed more than 300 putative proteins from over 80 species containing this amino acid motif, the Nudix box (Fig. 1). We have been systematically identifying and characterizing the enzymatic activities associated with these proteins, and we have found that almost all of the major substrates for these enzymes arenucleoside diphosphates linked to some other moiety, x, hence the acronym “Nudix.” The range of substrates acted on by various members of the family includes ribo- and deoxyribonucleoside triphosphates, nucleotide sugars, dinucleoside polyphosphates, NADH, and ADP-ribose. These substances are potentially toxic to the cell, signaling molecules, or metabolic intermediates whose concentrations require modulation during changes in the cell cycle or during periods of stress. We have suggested that the role of the Nudix hydrolases is to sanitize or modulate the accumulation of these metabolites (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Since the Nudix box is common to all of these enzymes, their specificity for the individual substrates must lie somewhere distal to the conserved region. In this paper, we describe the cloning and characterization of four ADP-ribose pyrophosphatases, and we identify a proline residue downstream of the conserved sequence common to members of this subfamily of Nudix hydrolases. Furthermore, we have observed that other recurring amino acids in this same region are predictive of two other subfamilies of the Nudix hydrolases, the dinucleoside polyphosphate pyrophosphatases and the NADH pyrophosphatases. We also demonstrate that ADP-ribose pyrophosphatase activity may play a role in tellurite resistance, since overexpression of this enzyme markedly increases the survival of cultures of Escherichia coli exposed to this toxic metalloid oxyanion. Primers were obtained from Integrated DNA Technologies (Coralville, IA). Biochemicals and enzymes were obtained from Sigma unless otherwise noted. Calf intestinal alkaline phosphatase was from Stratagene, and enzymes used in standard cloning procedures were from Life Technologies, Inc. and U.S. Biochemical Corp. E. coli strain MG1655 was kindly provided by Dr. Frederick R. Blattner (University of Wisconsin), and strains ofSaccharomyces cerevisiae, Bacillus subtilis, Hemophilus influenzae, and E. coli BL21 (DE3) were departmental stocks. Genes of interest were amplified from genomic DNA with forward primers incorporating an NdeI site and reverse primers incorporating a BamHI site. The insert was prepared by digestion with NdeI and BamHI followed by gel purification and it was ligated with the corresponding restriction sites of pET11b under control of the T7 lac promoter for expression. The cloned genes, with their accession numbers in parentheses are as follows: ysa1, S. cerevisiae(Q09176); orf209, E. coli (P36651);yqkg, B. subtilis (P54570); hi0398, H. influenzae (AAC22057). The plasmid constructs are designated pYSA1, pOrf209, pYQKG, and pHI0398, respectively. BL21 (DE3) cells containing the respective plasmid were grown at 37 °C in LB broth on a shaker to an A600 of about 0.6 and induced by the addition of isopropyl-β-d-thiogalactopyranoside to a concentration of 1 mm. The cells were allowed to grow for an additional 3 h, harvested, washed by suspension in isotonic saline, and centrifuged in preweighed centrifuge tubes, and the packed cells were stored at −80 °C. A summary of the steps involved in the purification of each of the enzymes follows. Cells were suspended in 3 volumes of 50 mmTris, pH 7.5, 1 mm EDTA (buffer A) supplemented with 0.1 mm dithiothreitol and 30% glycerol and disrupted in a French press. Glycerol was absolutely necessary for stabilization of the enzymatic activity throughout the purification procedure for YSA1. The protein was adjusted to 10 mg/ml, and nucleic acids were precipitated by adding streptomycin sulfate to a concentration of 1%. Ammonium sulfate was added to a final concentration of 50% saturation, and the precipitate was discarded after centrifugation. The supernatant was dialyzed and chromatographed on DEAE-Sepharose, and active fractions were pooled, dialyzed, and chromatographed on a hydroxyl apatite column. Cells were extracted as above in buffer A containing 1 mm EDTA and treated with streptomycin sulfate. A 30–60% ammonium sulfate fraction of the streptomycin supernatant was chromatographed on a gel filtration column (Sephadex G-100), and the active fractions were pooled, concentrated by precipitation in 80% ammonium sulfate, dialyzed, and chromatographed on DEAE-Sepharose. The purification of these two enzymes was considerably simplified, because almost all of the expressed protein leaked out of the frozen and thawed cells merely by washing them in buffer A. Endogenous proteins remained within the cells, resulting in an extract highly enriched for the expressed enzyme. The YQKG and HI0398 enzymes were recovered in an essentially pure state (>90%) by precipitating them in 70 or 30% ammonium sulfate, respectively. Enzyme velocities were quantitated by measuring the conversion of a phosphatase insensitive substrate, ADP-ribose, to the phosphatase-sensitive products, AMP and ribose 5-phosphate. The liberated inorganic orthophosphate was measured by the procedure of Ames and Dubin (3Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar). The standard incubation mixture (50 μl) contained 50 mm Tris-Cl, pH 8.0, 2 mm MgCl2, 2 mm ADP-ribose, 0.2–2 milliunits of enzyme, and 2 units of alkaline intestinal phosphatase. After 15 min at 37 °C, the reaction was terminated by the addition of EDTA, and inorganic orthophosphate was measured. A unit of enzyme hydrolyzes 1 μmol of substrate/min under these conditions. Note that 2 mol of phosphate are liberated per mol of ADP-ribose hydrolyzed. The standard assay mixture (minus alkaline intestinal phosphatase) was scaled up, and at various time intervals aliquots were analyzed by paper electrophoresis (4Markham R. Smith J.D. Biochem. J. 1952; 52: 552-557Crossref PubMed Scopus (173) Google Scholar), and additional aliquots were used for the determination of inorganic orthophosphate in the presence and absence of added alkaline intestinal phosphatase. Induction of BL21 (DE3) cells transformed with the cloned genes (see “Methods”) led to the appearance of new protein bands corresponding to molecular weights calculated from the respective amino acid content. Fig.2 shows an SDS-polyacrylamide gel comparing induced cells containing the cloned genes with control cells containing the vector, pET11b, without the inserted genes. In each case, a well defined new band is visible. When extracts of the cells prepared as described under “Methods” were centrifuged and analyzed by gel electrophoresis, the bulk of the newly expressed protein was in the soluble fraction (data not shown). It is interesting that two of the expressed proteins HI0398 (from H. influenzae) and YQKG (from B. subtilis) were extracted without mechanically disrupting the frozen cells, leaving the bulk of the other proteins behind as mentioned under “Methods.” This is reminiscent of two other Nudix hydrolases, Orf17 dATPase (5O'Handley S.F. Frick D.N. Bullions L.F. Mildvan A.S. Bessman M.J. J. Biol. Chem. 1996; 271: 24649-24654Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and the IalA diadenosine tetraphosphate pyrophosphatase (6Conyers G.B. Bessman M.J. J. Biol. Chem. 1999; 274: 1203-1206Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), both of which may be extracted by washing previously frozen cells. At present, it is not apparent why these proteins behave differently from most of the other Nudix hydrolases expressed in E. coli. Fig. 1 also shows the highly purified proteins resulting from the protocol described under “Methods.” These fractions were used for characterization of the enzymes reported below. Our initial studies of this group of enzymes began with Orf209. Although we did not know its enzymatic activity, we were influenced by our earlier work (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar) indicating that all of the major substrates for the Nudix hydrolase family were derivatives of nucleoside diphosphates. Accordingly, we screened a number of candidates in this structural category and found that ADP-ribose was an excellent substrate for the enzyme. This is shown in Table I along with the three other enzymes included in this study. For comparison, the two additional ADP-ribose pyrophosphatases described in earlier works (7O'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 (68) Google Scholar,8Sheikh S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) are also reported in the table. The activities toward ADP-ribose are compared with rates with some naturally occurring nucleoside diphosphate derivatives known to be favored substrates for other members of the Nudix hydrolase family (8Sheikh S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In each case, ADP-ribose is the preferred substrate, although there is a wide variation in absolute specificities. For example, MJ1149 from the archaebacterium, Methanococcus jannaschii, has no significant activity toward any of the other substrates, whereas Orf186 from E. coli and H10398 from H. influenzae have substantial activities on NADH and GDP-mannose, respectively. However, more rigorous kinetic analyses would be required for each of the putative substrates if a more substantive interpretation of the relative rates is in order. Kinetic parameters for ADP-ribose are compared in TableII, and a broad distribution in some of the constants is noted. These comparative values, derived under standard assay conditions, should be interpreted with caution, because differences in the physiology and ecology of the individual entries could have large effects on the data. For example, we have shown that the Vmax of MJ1149 increases 15-fold when assayed at 75 °C (8Sheikh S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), raising the rate from 6.2 to 93 units/mg, and this temperature is still 10 °C below the normal habitat of the organism (9Jones W.J. Leigh J.A. Mayer F. Woese C.R. Wolfe R.S. Arch. Microbiol. 1983; 136: 254-261Crossref Scopus (419) Google Scholar).Table IRelative specificities of the ADP-ribose hydrolasesSubstrateEnzymeYSA1Orf209HI0398YQKGOrf186aThese data for Orf186 and MJ1149 are taken from refs.7 and 8 respectively.MJ1149aThese data for Orf186 and MJ1149 are taken from refs.7 and 8 respectively.%%%%%%ADP-ribose10010010010093100GDP-mannose9159<1<1<1NADH12915272<1Ap3AbAp3A is adenosine (5′)-triphospho(5′)-adenosine.<1<1<1<1100<1(d)NTPc(d)NTP represents all eight of the canonical (deoxy)ribonucleoside triphosphates.<1<1<1<1<1<1The activity of the enzymes was measured at 37 °C using the standard assay described under “Methods.” When the nucleoside triphosphates were tested as substrates, the alkaline phosphatase was replaced by yeast inorganic pyrophosphatase (37Bhatnagar S.K. Bullions Bessman M.J. J. Biol. Chem. Full Text PDF PubMed Google These data for Orf186 and MJ1149 are taken from 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 (68) Google and S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google is adenosine represents all eight of the canonical (deoxy)ribonucleoside in a new Table of the ADP-ribose standard assay described under “Methods” was used with ADP-ribose concentrations of mm to kinetic and calculated from a was calculated from active site per A unit of enzyme hydrolyzes 1 μmol of in a new The activity of the enzymes was measured at 37 °C using the standard assay described under “Methods.” When the nucleoside triphosphates were tested as substrates, the alkaline phosphatase was replaced by yeast inorganic pyrophosphatase (37Bhatnagar S.K. Bullions Bessman M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The standard assay described under “Methods” was used with ADP-ribose concentrations of mm to kinetic and calculated from a was calculated from active site per A unit of enzyme hydrolyzes 1 μmol of with most of the Nudix hydrolases the ADP-ribose pyrophosphatase subfamily members have alkaline pH from pH 8 to absolutely require a for activity, with at 2 mm the preferred at concentrations is as of the enzymes from this has of activity when is replaced by and this is to the previously for Orf186 (7O'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 (68) Google Scholar). of standard reaction alkaline scaled up, were analyzed as described under “Methods.” inorganic phosphate was during the of the The of substrate ADP-ribose was with the appearance of and inorganic orthophosphate was incubation of the with alkaline intestinal phosphatase. The of the reaction may be as ADP-ribose AMP ribose 5-phosphate. The of a nucleoside activity associated with the E. coli gene S.K. Bessman M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and also with its of S. Bullions Bessman M.J. Mol. Microbiol. PubMed Scopus Google Bessman M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) suggested that the region of amino acid in the two otherwise proteins the site of these two A BLAST (2Altschul S.F. Gish W. Meyers E.W. Lipman D.J. J. Mol. Biol. 1990; 203: 403-410Crossref Scopus (69694) Google Scholar) search at that time revealed other in from to Bullions Bessman M.J. Mol. Microbiol. PubMed Scopus Google PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar) suggested that the conserved sequence designate nucleoside However, work has revealed that the enzyme is of a large family of enzymes with substrates NADH, dinucleoside polyphosphates, and as shown in the ADP-ribose a S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the large of to the the sequence has been linked to the enzymatic activity and to For example, MJ1149 of has been designated in the 1996; 273: PubMed Scopus Google Scholar), whereas in it has nucleoside activity and is in a highly specific ADP-ribose pyrophosphatase (8Sheikh S. O'Handley S.F. Dunn C.A. Bessman M.J. J. Biol. Chem. 1998; 273: 20924-20928Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). a recent E. 1999; PubMed Scopus Google Scholar) the resistance of to its large number of genes it from stress. In of the genes has been a new enzyme, A. S. and J. to the enzyme. This the an amino acid array by enzymes, and the a specific has For this we introduced the Nudix to the family of enzymes the Nudix box sequence of amino the enzyme of this large Fig. 1 shows a J.D. 1996; PubMed Scopus Google Scholar) of a of putative enzymes containing the Nudix box in a recent search of the data banks using the BLAST (2Altschul S.F. Gish W. Meyers E.W. Lipman D.J. J. Mol. Biol. 1990; 203: 403-410Crossref Scopus (69694) Google Scholar). Fig. 1 70 entries from a of 300 putative proteins from 75 in this are of enzymes on the substrates mentioned above and almost some enzymes with Since the Nudix box is common to all of these proteins, the of specificity must be to the Nudix of our in and characterizing new members of this interesting family of enzymes is to of the subfamilies in to the enzymatic activity of the of characterized enzymes of with activities more A is an of the ADP-ribose pyrophosphatase subfamily a highly conserved 15 or 16 amino acids downstream of the of the Nudix The entries have all been and the entries are we predicted the activity of the H. and B. subtilis proteins their respective genes were cloned and and we have that a (Fig. 3 is also an ADP-ribose pyrophosphatase and J. H. are shown in Fig. and for diadenosine polyphosphate hydrolases and the NADH hydrolases, respectively. In the there is a conserved tyrosine amino acids downstream from the Nudix box, and in the there is a of eight amino acids in this region. When this acid sequence was there was known NADH pyrophosphatase with the Nudix We have and the activities of the two additional entries and and J. both of which have the enzymatic activity predicted from the downstream The for of the Nudix hydrolases, has been by D.J. Frick D.N. J. Bessman M.J. Mildvan A.S. PubMed Scopus Google Scholar), and it has a 2 its and nucleotide site. The amino acids the three mentioned above would all be in 2 if this structural were conserved in all of the Nudix hydrolases. studies on the of the ADP-ribose pyrophosphatase J. and that the is in this enzyme as The of two additional enzymes, the dATPase (5O'Handley S.F. Frick D.N. Bullions L.F. Mildvan A.S. Bessman M.J. J. Biol. Chem. 1996; 271: 24649-24654Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and the hydrolase D.N. Bessman M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), are also in the of the by and the by J. and A. S. that we should have into the of the in the Nudix hydrolase It is interesting to that of the enzymes, has both the conserved proline conserved tyrosine (see Fig. A and This well with the specificity of which is almost active on and ADP-ribose On the other these amino acid of activity at present, be as to the in identifying new members of the since not all of the enzymes the For example, two hydrolases from the 1999; PubMed Scopus Google Scholar) and S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) not have the conserved tyrosine of the hydrolases, and which also has substantial activity on NADH (see Table not have the acid predictive of this to our and to demonstrate its was provided by the trgB gene of R. sphaeroides, which has been shown to be a tellurite resistance S. Microbiol. PubMed Google Scholar). Fig. 3 shows that the Nudix box in the proline the protein as an ADP-ribose pyrophosphatase. this enzyme could the tellurite resistance we transformed E. coli with a containing the ADP-ribose pyrophosphatase gene from the jannaschii, because the enzyme from this organism is highly specific for ADP-ribose (see Table in Fig. demonstrate that the ADP-ribose pyrophosphatase gene increases resistance to tellurite. In Fig. it be that the transformed almost at a 50% survival rate for the there was survival at Fig. shows the of tellurite on cultures of the transformed and cells. this is a specific of the ADP-ribose pyrophosphatase gene and not a of the Nudix hydrolases is shown in Fig. of the other genes tested tellurite with the in tellurite resistance was a in ADP-ribose pyrophosphatase in extracts of the transformed cells compared with the A. Dunn and J. resistance has been used for in the of and is a genes found in and a Microbiol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our and that of these genes, designates an ADP-ribose ADP-ribose as a factor in tellurite Although the role by ADP-ribose in this is not these are an of the of protein from amino acid in identifying the activities of proteins involved in It not that ADP-ribose is involved or tellurite ADP-ribose from the of and proteins during the of metabolic a J. Mol. Biochem. Scholar) and also by the large of to and of the coli and cells, Scholar). of its ADP-ribose amino and in proteins Mol. Biochem. PubMed Scopus Google J. Mol. Biochem. PubMed Scopus Google Scholar), enzymes or to proteins for H. A. S. A. PubMed Scopus Google R. A. PubMed Scopus Google H. J. Biochem. J. 1996; PubMed Scopus Google Scholar) or to proteins the the accumulation of ADP-ribose has been in the by of when it was shown that the ADP-ribose pyrophosphatase A. PubMed Scopus Google Scholar). We have also that this the yeast enzyme described in this has also been reported that of of the major of ADP-ribose, the by of H. A. R. W. PubMed Scopus Google Scholar). These recent support the large of data ADP-ribose as a mentioned in the we have suggested that the members of the Nudix hydrolase family of enzymes common a conserved amino acid sequence and a specificity for nucleoside diphosphate derivatives and that of their is to sanitize the cell of potentially toxic The ADP-ribose pyrophosphatases described in this paper these three and this subfamily as a of the Nudix hydrolases. We are to and for with