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

Studies on the ADP-ribose Pyrophosphatase Subfamily of the Nudix Hydrolases and Tentative... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 45, Issue of November 5, pp. 32318 –32324, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Studies on the ADP-ribose Pyrophosphatase Subfamily of the Nudix Hydrolases and Tentative Identification of trgB, a Gene Associated with Tellurite Resistance* (Received for publication, June 21, 1999, and in revised form, July 22, 1999) Christopher A. Dunn, Suzanne F. O’Handley‡, David N. Frick§, and Maurice J. Bessman¶ From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218 Four Nudix hydrolase genes, ysa1 from Saccharomy- the cell cycle or during periods of stress. We have suggested that the role of the Nudix hydrolases is to sanitize or modulate ces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influen- the accumulation of these metabolites (1). Since the Nudix box zae were amplified, cloned into an expression vector, is common to all of these enzymes, their specificity for the and transformed into E. coli. The expressed proteins individual substrates must lie somewhere distal to the con- were purified and shown to belong to a subfamily of served region. In this paper, we describe the cloning and char- Nudix hydrolases active on ADP-ribose. Comparison acterization of four ADP-ribose pyrophosphatases, and we with other members of the subfamily revealed a con- identify a proline residue downstream of the conserved se- served proline 16 amino acid residues downstream of quence common to members of this subfamily of Nudix hydro- the Nudix box, common to all of the ADP-ribose pyro- lases. Furthermore, we have observed that other recurring phosphatase subfamily. In this same region, a conserved amino acids in this same region are predictive of two other tyrosine designates another subfamily, the diadenosine subfamilies of the Nudix hydrolases, the dinucleoside poly- polyphosphate pyrophosphatases, while an array of phosphate pyrophosphatases and the NADH pyrophosphatases. eight conserved amino acids is indicative of the NADH We also demonstrate that ADP-ribose pyrophosphatase ac- pyrophosphatases. On the basis of these classifications, tivity may play a role in tellurite resistance, since overexpres- the trgB gene, a tellurite resistance factor from sion of this enzyme markedly increases the survival of cultures Rhodobacter sphaeroides, was predicted to designate an of Escherichia coli exposed to this toxic metalloid oxyanion. ADP-ribose pyrophosphatase. In support of this hypoth- esis, a highly specific ADP-ribose pyrophosphatase gene EXPERIMENTAL PROCEDURES from the archaebacterium, Methanococcus jannaschii, Materials introduced into E. coli, increased the transformant’s tol- erance to potassium tellurite. Primers were obtained from Integrated DNA Technologies (Cor- alville, IA). Biochemicals and enzymes were obtained from Sigma un- less otherwise noted. Calf intestinal alkaline phosphatase was from Stratagene, and enzymes used in standard cloning procedures were from The Nudix hydrolases comprise a large family of proteins Life Technologies, Inc. and U.S. Biochemical Corp. E. coli strain MG1655 characterized by the highly conserved array of amino acids was kindly provided by Dr. Frederick R. Blattner (University of Wiscon- GX EX REUXEEXGU, where U represents a bulky, hydropho- 5 7 sin), and strains of Saccharomyces cerevisiae, Bacillus subtilis, He- bic, amino acid, usually Ile, Leu, or Val (1). A recent BLAST (2) mophilus influenzae, and E. coli BL21 (DE3) were departmental stocks. search of the sequence data banks has revealed more than 300 Cloning putative proteins from over 80 species containing this amino Genes of interest were amplified from genomic DNA with forward acid motif, the Nudix box (Fig. 1). We have been systematically primers incorporating an NdeI site and reverse primers incorporating a identifying and characterizing the enzymatic activities associ- BamHI site. The insert was prepared by digestion with NdeI and ated with these proteins, and we have found that almost all of BamHI followed by gel purification and it was ligated with the corre- the major substrates for these enzymes are nucleoside diphos- sponding restriction sites of pET11b under control of the T7 lac pro- phates linked to some other moiety, x, hence the acronym moter for expression. The cloned genes, with their accession numbers in parentheses are as follows: ysa1, S. cerevisiae (Q09176); orf209, E. coli “Nudix.” The range of substrates acted on by various members (P36651); yqkg, B. subtilis (P54570); hi0398, H. influenzae (AAC22057). of the family includes ribo- and deoxyribonucleoside triphos- The plasmid constructs are designated pYSA1, pOrf209, pYQKG, and phates, nucleotide sugars, dinucleoside polyphosphates, pHI0398, respectively. NADH, and ADP-ribose. These substances are potentially toxic Expression and Purification of the Enzymes to the cell, signaling molecules, or metabolic intermediates whose concentrations require modulation during changes in BL21 (DE3) cells containing the respective plasmid were grown at 37 °C in LB broth on a shaker to an A of about 0.6 and induced by the b-D-thiogalactopyranoside to a concentration of 1 addition of isopropyl- * This work was supported by National Institutes of Health Grant mM. The cells were allowed to grow for an additional 3 h, harvested, GM 18649. This is publication 1521 from the McCollum-Pratt Institute. washed by suspension in isotonic saline, and centrifuged in preweighed The costs of publication of this article were defrayed in part by the centrifuge tubes, and the packed cells were stored at 280 °C. A sum- payment of page charges. This article must therefore be hereby marked mary of the steps involved in the purification of each of the enzymes “advertisement” in accordance with 18 U.S.C. Section 1734 solely to follows. indicate this fact. YSA1—Cells were suspended in 3 volumes of 50 mM Tris, pH 7.5, 1 ‡ Present address: Dept. of Chemistry, University of Richmond, Rich- mM EDTA (buffer A) supplemented with 0.1 mM dithiothreitol and 30% mond, VA 23173. glycerol and disrupted in a French press. Glycerol was absolutely nec- § Present address: Dept. of Biological Chemistry, Harvard Medical essary for stabilization of the enzymatic activity throughout the puri- School, Boston, MA 02115. ¶ fication procedure for YSA1. The protein was adjusted to 10 mg/ml, and To whom correspondence should be addressed. Tel.: 410-516-7316; Fax: 410-516-5213; E-mail: [email protected]. nucleic acids were precipitated by adding streptomycin sulfate to a 32318 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32319 concentration of 1%. Ammonium sulfate was added to a final concen- were pooled, concentrated by precipitation in 80% ammonium sulfate, tration of 50% saturation, and the precipitate was discarded after dialyzed, and chromatographed on DEAE-Sepharose. centrifugation. The supernatant was dialyzed and chromatographed on YQKG and HI0398 —The purification of these two enzymes was DEAE-Sepharose, and active fractions were pooled, dialyzed, and chro- considerably simplified, because almost all of the expressed protein matographed on a hydroxyl apatite column. leaked out of the frozen and thawed cells merely by washing them in Orf209 —Cells were extracted as above in buffer A containing 1 mM buffer A. Endogenous proteins remained within the cells, resulting in EDTA and treated with streptomycin sulfate. A 30 – 60% ammonium an extract highly enriched for the expressed enzyme. The YQKG and sulfate fraction of the streptomycin supernatant was chromatographed HI0398 enzymes were recovered in an essentially pure state (.90%) by on a gel filtration column (Sephadex G-100), and the active fractions precipitating them in 70 or 30% ammonium sulfate, respectively. FIG.1. Representatives of the Nudix hydrolase family. A recent BLAST search (2) revealed more than 300 putative members of the Nudix hydrolase family from over 70 species. Shown is a sample of 70 entries from 43 species illustrating the highly conserved Nudix signature sequence. 32320 ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases Methods Enzyme Assay 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 inor- ganic orthophosphate was measured by the procedure of Ames and Dubin (3). The standard incubation mixture (50 ml) contained 50 mM Tris-Cl, pH 8.0, 2 mM MgCl ,2mM 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 mmol of substrate/min under these conditions. Note that 2 mol of phos- phate are liberated per mol of ADP-ribose hydrolyzed. Product Determination FIG.2. Expression and purification of the ADP-ribose pyro- phosphatases. A polyacrylamide gel (15%) containing 1% SDS and The standard assay mixture (minus alkaline intestinal phosphatase) stained with Coomassie Blue contained, in lane 1, reference proteins was scaled up, and at various time intervals aliquots were analyzed by with the following molecular masses: 66, 45, 36, 29, 24, 20, 14. 2, and 6.5 paper electrophoresis (4), and additional aliquots were used for the kDa. Lanes 2, 3, 5, 7, and 9 contained approximately 7.5 mg of crude determination of inorganic orthophosphate in the presence and absence extracts from cells containing pET 11 alone or containing pYSA1, of added alkaline intestinal phosphatase. pOrf209, pYQKG, or pHI0398, respectively. Lanes 4, 6, 8, and 10 are the purified fractions of each of these enzymes, adjusted to contain approx- RESULTS AND DISCUSSION imately the same number of enzyme units as in the respective crude extract. Expression and Purification of Proteins—Induction of BL21 (DE3) cells transformed with the cloned genes (see “Methods”) TABLE I led to the appearance of new protein bands corresponding to Relative specificities of the ADP-ribose hydrolases molecular weights calculated from the respective amino acid The activity of the enzymes was measured at 37 °C using the stand- content. Fig. 2 shows an SDS-polyacrylamide gel comparing ard assay described under “Methods.” When the nucleoside triphos- induced cells containing the cloned genes with control cells phates were tested as substrates, the alkaline phosphatase was re- placed by yeast inorganic pyrophosphatase (37). containing the vector, pET11b, without the inserted genes. In each case, a well defined new band is visible. When extracts of Enzyme Substrate the cells prepared as described under “Methods” were centri- a a YSA1 Orf209 HI0398 YQKG Orf186 MJ1149 fuged and analyzed by gel electrophoresis, the bulk of the %% % % % % newly expressed protein was in the soluble fraction (data not ADP-ribose 100 100 100 100 93 100 shown). It is interesting that two of the expressed proteins GDP-mannose 9 1 59 ,1 ,1 ,1 HI0398 (from H. influenzae) and YQKG (from B. subtilis) were NADH 12 9 15 2 72 ,1 extracted without mechanically disrupting the frozen cells, Ap A ,1 ,1 ,1 ,1 100 ,1 (d)NTP ,1 ,1 ,1 ,1 ,1 ,1 leaving the bulk of the other proteins behind as mentioned under “Methods.” This is reminiscent of two other Nudix hy- These data for Orf186 and MJ1149 are taken from refs. 7 and 8 respectively. drolases, Orf17 dATPase (5) and the IalA diadenosine tetra- Ap A is adenosine (59)-triphospho(59)-adenosine. phosphate pyrophosphatase (6), both of which may be extracted (d)NTP represents all eight of the canonical (deoxy)ribonucleoside by washing previously frozen cells. At present, it is not appar- triphosphates. ent why these proteins behave differently from most of the other Nudix hydrolases expressed in E. coli. Fig. 1 also shows TABLE II the highly purified proteins resulting from the protocol de- Kinetic analysis of the ADP-ribose hydrolases scribed under “Methods.” These fractions were used for char- The standard assay described under “Methods” was used with ADP- acterization of the enzymes reported below. ribose concentrations of 0.05– 4.0 mM to determine kinetic constants. K and V were calculated from a nonlinear regression analysis. K Enzymatic Activities—Our initial studies of this group of m max cat was calculated from V assuming one active site per monomer. A unit max enzymes began with Orf209. Although we did not know its of enzyme hydrolyzes 1 mmol of substrate/min. enzymatic activity, we were influenced by our earlier work (1) Enzyme V K K K /K indicating that all of the major substrates for the Nudix hydro- max cat m cat m 21 21 21 21 lase family were derivatives of nucleoside diphosphates. Ac- units mg s mMM s cordingly, we screened a number of candidates in this struc- YSA1 182.3 6 5.5 79.2 6 2.4 0.040 6 0.009 1.98 3 10 tural category and found that ADP-ribose was an excellent Orf209 297.5 6 5.1 117.3 6 2.0 0.067 6 0.007 1.75 3 10 YQKG 185.2 6 4.0 64.8 6 1.4 0.114 6 0.013 5.68 3 10 substrate for the enzyme. This is shown in Table I along with HI0398 22.6 6 1.1 9.2 6 0.4 0.063 6 0.014 1.46 3 10 the three other enzymes included in this study. For compari- Orf186 28.0 6 1.0 9.9 6 0.4 0.220 6 0.027 4.5 3 10 son, the two additional ADP-ribose pyrophosphatases de- 3 MJ1149 6.2 6 0.3 1.9 6 0.08 0.340 6 0.050 5.82 3 10 scribed in earlier works (7, 8) are also reported in the table. The activities toward ADP-ribose are compared with rates with some naturally occurring nucleoside diphosphate derivatives rates is in order. Kinetic parameters for ADP-ribose are com- known to be favored substrates for other members of the Nudix pared in Table II, and a broad distribution in some of the hydrolase family (8). In each case, ADP-ribose is the preferred constants is noted. These comparative values, derived under substrate, although there is a wide variation in absolute spec- standard assay conditions, should be interpreted with caution, ificities. For example, MJ1149 from the archaebacterium, because differences in the physiology and ecology of the indi- Methanococcus jannaschii, has no significant activity toward vidual entries could have large effects on the data. For exam- any of the other substrates, whereas Orf186 from E. coli and ple, we have shown that the V of MJ1149 increases 15-fold max H10398 from H. influenzae have substantial activities on when assayed at 75 °C (8), raising the rate from 6.2 to 93 NADH and GDP-mannose, respectively. However, more rigor- units/mg, and this temperature is still 10 °C below the normal ous kinetic analyses would be required for each of the putative habitat of the organism (9). substrates if a more substantive interpretation of the relative Other Properties of the Enzymes—As with most of the Nudix ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32321 FIG.3. Subfamilies of the Nudix hy- drolases. In each group, the amino acids constituting the Nudix box are in boldface type along with the downstream amino acid(s) distinguishing each subfamily. The check marks indicate proteins whose enzymatic activities have been positively identified. The source for Orf176 is J. D. Walsh and M. J. Bessman (unpublished results). hydrolases studied so far, the ADP-ribose pyrophosphatase Products of the Reaction—Aliquots of standard reaction mix- subfamily members have distinctly alkaline pH optima, rang- tures (omitting alkaline phosphatase), appropriately scaled up, ing from pH 8 to 9. All absolutely require a divalent cation for were analyzed as described under “Methods.” No inorganic activity, with Mg at approximately 2 mM the preferred metal. phosphate was formed during the course of the reactions. The Mn at equal concentrations is 10 –20% as effective. One of disappearance of substrate ADP-ribose was coincident with the the enzymes from this study, Orf209, has approximately 40% of appearance of AMP, and inorganic orthophosphate was re- 21 21 maximal activity when Mg is replaced by Zn , and this is leased upon incubation of the products with alkaline intestinal similar to the results seen previously for Orf186 (7). phosphatase. The course of the reaction may be written as 32322 ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases ADP-ribose 1 H O 3 AMP 1 ribose 5-phosphate. box, and in the latter, there is a consensus of eight amino acids in this region. When this 8-amino acid consensus sequence was Identification of Nudix Hydrolase Subfamilies—The discov- ery of a nucleoside triphosphatase activity associated with the first uncovered, there was only one known NADH pyrophos- E. coli mutT1 mutator gene (10) and also with its ortholog, phatase with the Nudix motif. We have recently cloned, ex- mutX,of S. pneumoniae (11, 12) suggested that the small pressed, purified, and determined the activities of the two region of amino acid identity in the two otherwise dissimilar additional entries XGLO67w (S. cerevisiae), and F13H10 (C. proteins comprised the catalytic site of these two enzymes. A elegans), both of which have the enzymatic activity predicted BLAST (2) search at that time revealed 13 other open reading from the downstream signals. The three-dimensional solution frames present in organisms ranging from viruses to humans structure for one of the Nudix hydrolases, MutT, has been (11, 13), and Koonin (13) suggested that the conserved MutT solved by NMR spectroscopy (17), and it has a unique, loop signature sequence might designate nucleoside triphosphate 1-helix-loop 2 motif encompassing its catalytic and nucleotide pyrophosphohydrolase activity. However, subsequent work has binding site. The characteristic amino acids distinguishing the revealed that the MutT enzyme is only one member of a large three families mentioned above would all be in loop 2 if this family of different enzymes with different substrates including structural feature were conserved in all of the Nudix hydro- sugar nucleotides, NADH, dinucleoside polyphosphates, and as lases. Preliminary studies on the crystal structure of the shown in the present work, ADP-ribose (for a review, see Ref. Orf209 ADP-ribose pyrophosphatase suggest that the loop- 8). Despite the large body of evidence to the contrary, the MutT helix-loop motif is present in this enzyme as well. The three- signature sequence (GX dimensional structures of two additional enzymes, Orf17, the EX REUXEEXGU) has been errone- 5 7 ously linked to the MutT enzymatic activity and to anti- dATPase (5), and Orf1.9, the GDP-mannose hydrolase (18), are mutagenesis. For example, MJ1149 of M. jannaschii has been also in the process of being solved, the former by x-ray crystal- 4 5 designated MutT in the TIGR sequencing project (14), whereas lography and the latter by NMR spectroscopy, so that we in reality it has neither nucleoside triphosphatase nor anti- should soon have insight into the generality of the loop-helix- mutagenic activity and is in fact a highly specific ADP-ribose loop motif in the Nudix hydrolase family. It is interesting to pyrophosphatase (8). Also, a recent report (15) attributes the note that one of the enzymes, Orf186, has both the conserved extreme radiation resistance of Deinococcus radiodurans to its proline and the conserved tyrosine (see Fig. 3, A and B). This large number of MutT genes insulating it from oxidative stress. correlates well with the specificity of Orf186, which is almost In fact, only one of the genes has been identified so far, gdr8, equally active on Ap A and ADP-ribose (Table I). On the other designating a new enzyme, UDP-glucose pyrophosphatase, hand, these amino acid predictors of activity should, at present, unrelated to the MutT enzyme. This ambiguity between the be viewed only as clues to narrow down the possibilities in “MutT motif,” an amino acid array shared by several different identifying new members of the family, since not all of the enzymes, and the “MutT enzyme,” connoting a specific physio- enzymes fit the present patterns. For example, two Ap A hy- logical function, has caused considerable confusion. For this drolases from the yeasts Schizosaccharomyces pombe (19) and reason, we introduced the acronym, Nudix hydrolase, to define S. cerevisiae (20) do not have the conserved tyrosine of the the family of different enzymes sharing the Nudix box signa- Ap A hydrolases, and Orf186, which also has substantial ac- ture sequence of amino acids, the MutT enzyme being only one tivity on NADH (see Table I) does not have the 8-amino acid member of this large family. Fig. 1 shows a Clustal (16) align- consensus predictive of this activity. ment of a partial list of putative enzymes containing the Nudix ADP-ribose Pyrophosphatase and Tellurite Resistance—An box (MutT motif) uncovered in a recent search of the data opportunity to test our classification scheme and to demon- banks using the BLAST program (2). Fig. 1 contains 70 entries strate its potential utility was provided by the trgB gene of R. selected from a total of 300 putative proteins from 75 different sphaeroides, which has been shown to be a tellurite resistance species. Sequestered in this list are different families of en- determinant (21). Fig. 3A, line 7, shows that TRGB contains zymes acting on the substrates mentioned above and almost the Nudix box and, in addition, the signal proline tentatively certainly some enzymes with novel, undiscovered activities. categorizing the protein as an ADP-ribose pyrophosphatase. To Since the Nudix box is common to all of these proteins, the test whether this enzyme could confer the tellurite resistance determinant(s) of specificity must be supplementary to the phenotype, we transformed E. coli with a plasmid, pTRC, con- Nudix signature sequence. One of our objectives in discovering taining the ADP-ribose pyrophosphatase gene (MJ1149) from and characterizing new members of this interesting family of the archaebacterium M. jannaschii, because the enzyme from enzymes is to recognize distinguishing features of the subfam- this organism is highly specific for ADP-ribose (see Table I). ilies in order to predict the enzymatic activity of undetermined The graphs in Fig. 4 clearly demonstrate that the ADP-ribose entries. As the collection of characterized enzymes grows, pyrophosphatase gene increases resistance to tellurite. In Fig. alignments of those with similar activities become more reveal- 4A, it can be seen that the transformed culture retained almost ing. Fig. 3A is an alignment of the ADP-ribose pyrophosphatase 100% viability at a 50% survival rate for the parent culture. subfamily showing a highly conserved proline, 15 or 16 amino Likewise, there was approximately 60% survival versus 100% acids downstream of the terminal glycine of the Nudix box. The killing, at 0.5 mg/ml K TeO . Fig. 4B shows the differential 2 3 checked entries have all been identified, and the remaining effect of tellurite on growing cultures of the transformed and entries are predicted. Actually, we predicted the activity of the parent cells. That this is a specific effect of the ADP-ribose H. influenza and B. subtilis proteins before their respective pyrophosphatase gene and not a general property of the Nudix genes were cloned and expressed, and we have recently learned hydrolases is shown in Fig. 4C. None of the other genes tested that a human EST (Fig. 3A, line 17) is also an ADP-ribose confer tellurite resistance. Commensurate with the increase in pyrophosphatase Similar alignments are shown in Fig. 3, B tellurite resistance was a 5-fold increase in ADP-ribose pyro- and C, for diadenosine polyphosphate hydrolases and the phosphatase in crude extracts of the transformed cells com- NADH hydrolases, respectively. In the former, there is a con- pared with the parent culture. served tyrosine 16 –18 amino acids downstream from the Nudix W-L. Xu and M. J. Bessman, unpublished results. S. Gabelli, M. J. Bessman, and L. M. Amzel, unpublished results. 1 5 C. A. Dunn, S. Desai, and M. J. Bessman, unpublished results. P. Leggett, M. J. Bessman, and A. S. Mildvan, unpublished results. 2 6 H. Yang, M. Slupska, and J. H. Miller, personal communication. C. A. Dunn and M. J. Bessman, unpublished observations. ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32323 Tellurite resistance has been used for many years in the differential diagnosis of pathological microorganisms and is a complex process implicating many genes found in prokaryotes and eukaryotes (for a review, see Ref. 22). Our experiments and classification scheme strongly suggest that one of these genes, trgB, designates an ADP-ribose pyrophosphatase, implicating ADP-ribose itself as a factor in tellurite sensitivity. Although the role played by ADP-ribose in this process is not known, these experiments are an example of how functional genomics, the prediction of protein function from amino acid sequence, can aid in identifying the activities of unknown proteins in- volved in physiological processes. It is, perhaps, not surprising that ADP-ribose is directly involved in, or potentiates, tellurite toxicity. Cellular ADP- ribose arises from the hydrolysis of mono- and poly(A)DP- ribosylated proteins during the regulation of metabolic pro- cesses (for a review, see Ref. 23) and also by the large scale turnover of NAD , amounting to approximately 30 and 90% of the total NAD synthesis in E. coli and HeLa cells, respectively (24). Because of its free aldehydic group, ADP-ribose can deri- vatize terminal amino groups, lysines, and cysteines in pro- teins nonenzymatically (25, 26), thereby inactivating enzymes or leading to proteins targeted for apoptosis (27–29) or to non- specifically tagged proteins confusing the ADP-ribosylation recognition system. Recently, the accumulation of ADP-ribose has been implicated in the liver damage caused by high levels of acetaminophen (N-acetyl-p-aminophenol, Tylenol ) when it was shown that the acetaminophen metabolite, N-acetyl-p- benzoquinonimine, inhibits rat liver ADP-ribose pyrophos- phatase (30). We have also seen that this derivative inhibits YSA1, the yeast enzyme described in this paper. It has also been reported that inhibitors of poly(ADP-ribose) polymerase, one of the major sources of cellular ADP-ribose, prevent the liver damage caused by overdoses of acetaminophen (31). These recent experiments support the large body of data implicating free ADP-ribose as a cytotoxic agent. As mentioned in the Introduction, we have suggested that the members of the Nudix hydrolase family of enzymes share common features including a conserved amino acid signature sequence and a specificity for nucleoside diphosphate deriva- tives and that one of their physiological functions is to sanitize the cell of potentially toxic metabolites. The ubiquitous ADP- ribose pyrophosphatases described in this paper meet these three criteria and qualify this subfamily as a bona fide member of the Nudix hydrolases. Acknowledgments—We are indebted to Drs. Samuel Kaplan and Mark Gomelsky for help with trgB. 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Studies on the ADP-ribose Pyrophosphatase Subfamily of the Nudix Hydrolases and Tentative Identification of trgB, a Gene Associated with Tellurite Resistance

Dunn, Christopher A.; O'Handley, Suzanne F.; Frick, David N.; Bessman, Maurice J.
Journal of Biological Chemistry – Nov 1, 1999
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 45, Issue of November 5, pp. 32318 –32324, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Studies on the ADP-ribose Pyrophosphatase Subfamily of the Nudix Hydrolases and Tentative Identification of trgB, a Gene Associated with Tellurite Resistance* (Received for publication, June 21, 1999, and in revised form, July 22, 1999) Christopher A. Dunn, Suzanne F. O’Handley‡, David N. Frick§, and Maurice J. Bessman¶ From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218 Four Nudix hydrolase genes, ysa1 from Saccharomy- the cell cycle or during periods of stress. We have suggested that the role of the Nudix hydrolases is to sanitize or modulate ces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influen- the accumulation of these metabolites (1). Since the Nudix box zae were amplified, cloned into an expression vector, is common to all of these enzymes, their specificity for the and transformed into E. coli. The expressed proteins individual substrates must lie somewhere distal to the con- were purified and shown to belong to a subfamily of served region. In this paper, we describe the cloning and char- Nudix hydrolases active on ADP-ribose. Comparison acterization of four ADP-ribose pyrophosphatases, and we with other members of the subfamily revealed a con- identify a proline residue downstream of the conserved se- served proline 16 amino acid residues downstream of quence common to members of this subfamily of Nudix hydro- the Nudix box, common to all of the ADP-ribose pyro- lases. Furthermore, we have observed that other recurring phosphatase subfamily. In this same region, a conserved amino acids in this same region are predictive of two other tyrosine designates another subfamily, the diadenosine subfamilies of the Nudix hydrolases, the dinucleoside poly- polyphosphate pyrophosphatases, while an array of phosphate pyrophosphatases and the NADH pyrophosphatases. eight conserved amino acids is indicative of the NADH We also demonstrate that ADP-ribose pyrophosphatase ac- pyrophosphatases. On the basis of these classifications, tivity may play a role in tellurite resistance, since overexpres- the trgB gene, a tellurite resistance factor from sion of this enzyme markedly increases the survival of cultures Rhodobacter sphaeroides, was predicted to designate an of Escherichia coli exposed to this toxic metalloid oxyanion. ADP-ribose pyrophosphatase. In support of this hypoth- esis, a highly specific ADP-ribose pyrophosphatase gene EXPERIMENTAL PROCEDURES from the archaebacterium, Methanococcus jannaschii, Materials introduced into E. coli, increased the transformant’s tol- erance to potassium tellurite. Primers were obtained from Integrated DNA Technologies (Cor- alville, IA). Biochemicals and enzymes were obtained from Sigma un- less otherwise noted. Calf intestinal alkaline phosphatase was from Stratagene, and enzymes used in standard cloning procedures were from The Nudix hydrolases comprise a large family of proteins Life Technologies, Inc. and U.S. Biochemical Corp. E. coli strain MG1655 characterized by the highly conserved array of amino acids was kindly provided by Dr. Frederick R. Blattner (University of Wiscon- GX EX REUXEEXGU, where U represents a bulky, hydropho- 5 7 sin), and strains of Saccharomyces cerevisiae, Bacillus subtilis, He- bic, amino acid, usually Ile, Leu, or Val (1). A recent BLAST (2) mophilus influenzae, and E. coli BL21 (DE3) were departmental stocks. search of the sequence data banks has revealed more than 300 Cloning putative proteins from over 80 species containing this amino Genes of interest were amplified from genomic DNA with forward acid motif, the Nudix box (Fig. 1). We have been systematically primers incorporating an NdeI site and reverse primers incorporating a identifying and characterizing the enzymatic activities associ- BamHI site. The insert was prepared by digestion with NdeI and ated with these proteins, and we have found that almost all of BamHI followed by gel purification and it was ligated with the corre- the major substrates for these enzymes are nucleoside diphos- sponding restriction sites of pET11b under control of the T7 lac pro- phates linked to some other moiety, x, hence the acronym moter for expression. The cloned genes, with their accession numbers in parentheses are as follows: ysa1, S. cerevisiae (Q09176); orf209, E. coli “Nudix.” The range of substrates acted on by various members (P36651); yqkg, B. subtilis (P54570); hi0398, H. influenzae (AAC22057). of the family includes ribo- and deoxyribonucleoside triphos- The plasmid constructs are designated pYSA1, pOrf209, pYQKG, and phates, nucleotide sugars, dinucleoside polyphosphates, pHI0398, respectively. NADH, and ADP-ribose. These substances are potentially toxic Expression and Purification of the Enzymes to the cell, signaling molecules, or metabolic intermediates whose concentrations require modulation during changes in BL21 (DE3) cells containing the respective plasmid were grown at 37 °C in LB broth on a shaker to an A of about 0.6 and induced by the b-D-thiogalactopyranoside to a concentration of 1 addition of isopropyl- * This work was supported by National Institutes of Health Grant mM. The cells were allowed to grow for an additional 3 h, harvested, GM 18649. This is publication 1521 from the McCollum-Pratt Institute. washed by suspension in isotonic saline, and centrifuged in preweighed The costs of publication of this article were defrayed in part by the centrifuge tubes, and the packed cells were stored at 280 °C. A sum- payment of page charges. This article must therefore be hereby marked mary of the steps involved in the purification of each of the enzymes “advertisement” in accordance with 18 U.S.C. Section 1734 solely to follows. indicate this fact. YSA1—Cells were suspended in 3 volumes of 50 mM Tris, pH 7.5, 1 ‡ Present address: Dept. of Chemistry, University of Richmond, Rich- mM EDTA (buffer A) supplemented with 0.1 mM dithiothreitol and 30% mond, VA 23173. glycerol and disrupted in a French press. Glycerol was absolutely nec- § Present address: Dept. of Biological Chemistry, Harvard Medical essary for stabilization of the enzymatic activity throughout the puri- School, Boston, MA 02115. ¶ fication procedure for YSA1. The protein was adjusted to 10 mg/ml, and To whom correspondence should be addressed. Tel.: 410-516-7316; Fax: 410-516-5213; E-mail: [email protected]. nucleic acids were precipitated by adding streptomycin sulfate to a 32318 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32319 concentration of 1%. Ammonium sulfate was added to a final concen- were pooled, concentrated by precipitation in 80% ammonium sulfate, tration of 50% saturation, and the precipitate was discarded after dialyzed, and chromatographed on DEAE-Sepharose. centrifugation. The supernatant was dialyzed and chromatographed on YQKG and HI0398 —The purification of these two enzymes was DEAE-Sepharose, and active fractions were pooled, dialyzed, and chro- considerably simplified, because almost all of the expressed protein matographed on a hydroxyl apatite column. leaked out of the frozen and thawed cells merely by washing them in Orf209 —Cells were extracted as above in buffer A containing 1 mM buffer A. Endogenous proteins remained within the cells, resulting in EDTA and treated with streptomycin sulfate. A 30 – 60% ammonium an extract highly enriched for the expressed enzyme. The YQKG and sulfate fraction of the streptomycin supernatant was chromatographed HI0398 enzymes were recovered in an essentially pure state (.90%) by on a gel filtration column (Sephadex G-100), and the active fractions precipitating them in 70 or 30% ammonium sulfate, respectively. FIG.1. Representatives of the Nudix hydrolase family. A recent BLAST search (2) revealed more than 300 putative members of the Nudix hydrolase family from over 70 species. Shown is a sample of 70 entries from 43 species illustrating the highly conserved Nudix signature sequence. 32320 ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases Methods Enzyme Assay 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 inor- ganic orthophosphate was measured by the procedure of Ames and Dubin (3). The standard incubation mixture (50 ml) contained 50 mM Tris-Cl, pH 8.0, 2 mM MgCl ,2mM 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 mmol of substrate/min under these conditions. Note that 2 mol of phos- phate are liberated per mol of ADP-ribose hydrolyzed. Product Determination FIG.2. Expression and purification of the ADP-ribose pyro- phosphatases. A polyacrylamide gel (15%) containing 1% SDS and The standard assay mixture (minus alkaline intestinal phosphatase) stained with Coomassie Blue contained, in lane 1, reference proteins was scaled up, and at various time intervals aliquots were analyzed by with the following molecular masses: 66, 45, 36, 29, 24, 20, 14. 2, and 6.5 paper electrophoresis (4), and additional aliquots were used for the kDa. Lanes 2, 3, 5, 7, and 9 contained approximately 7.5 mg of crude determination of inorganic orthophosphate in the presence and absence extracts from cells containing pET 11 alone or containing pYSA1, of added alkaline intestinal phosphatase. pOrf209, pYQKG, or pHI0398, respectively. Lanes 4, 6, 8, and 10 are the purified fractions of each of these enzymes, adjusted to contain approx- RESULTS AND DISCUSSION imately the same number of enzyme units as in the respective crude extract. Expression and Purification of Proteins—Induction of BL21 (DE3) cells transformed with the cloned genes (see “Methods”) TABLE I led to the appearance of new protein bands corresponding to Relative specificities of the ADP-ribose hydrolases molecular weights calculated from the respective amino acid The activity of the enzymes was measured at 37 °C using the stand- content. Fig. 2 shows an SDS-polyacrylamide gel comparing ard assay described under “Methods.” When the nucleoside triphos- induced cells containing the cloned genes with control cells phates were tested as substrates, the alkaline phosphatase was re- placed by yeast inorganic pyrophosphatase (37). containing the vector, pET11b, without the inserted genes. In each case, a well defined new band is visible. When extracts of Enzyme Substrate the cells prepared as described under “Methods” were centri- a a YSA1 Orf209 HI0398 YQKG Orf186 MJ1149 fuged and analyzed by gel electrophoresis, the bulk of the %% % % % % newly expressed protein was in the soluble fraction (data not ADP-ribose 100 100 100 100 93 100 shown). It is interesting that two of the expressed proteins GDP-mannose 9 1 59 ,1 ,1 ,1 HI0398 (from H. influenzae) and YQKG (from B. subtilis) were NADH 12 9 15 2 72 ,1 extracted without mechanically disrupting the frozen cells, Ap A ,1 ,1 ,1 ,1 100 ,1 (d)NTP ,1 ,1 ,1 ,1 ,1 ,1 leaving the bulk of the other proteins behind as mentioned under “Methods.” This is reminiscent of two other Nudix hy- These data for Orf186 and MJ1149 are taken from refs. 7 and 8 respectively. drolases, Orf17 dATPase (5) and the IalA diadenosine tetra- Ap A is adenosine (59)-triphospho(59)-adenosine. phosphate pyrophosphatase (6), both of which may be extracted (d)NTP represents all eight of the canonical (deoxy)ribonucleoside by washing previously frozen cells. At present, it is not appar- triphosphates. ent why these proteins behave differently from most of the other Nudix hydrolases expressed in E. coli. Fig. 1 also shows TABLE II the highly purified proteins resulting from the protocol de- Kinetic analysis of the ADP-ribose hydrolases scribed under “Methods.” These fractions were used for char- The standard assay described under “Methods” was used with ADP- acterization of the enzymes reported below. ribose concentrations of 0.05– 4.0 mM to determine kinetic constants. K and V were calculated from a nonlinear regression analysis. K Enzymatic Activities—Our initial studies of this group of m max cat was calculated from V assuming one active site per monomer. A unit max enzymes began with Orf209. Although we did not know its of enzyme hydrolyzes 1 mmol of substrate/min. enzymatic activity, we were influenced by our earlier work (1) Enzyme V K K K /K indicating that all of the major substrates for the Nudix hydro- max cat m cat m 21 21 21 21 lase family were derivatives of nucleoside diphosphates. Ac- units mg s mMM s cordingly, we screened a number of candidates in this struc- YSA1 182.3 6 5.5 79.2 6 2.4 0.040 6 0.009 1.98 3 10 tural category and found that ADP-ribose was an excellent Orf209 297.5 6 5.1 117.3 6 2.0 0.067 6 0.007 1.75 3 10 YQKG 185.2 6 4.0 64.8 6 1.4 0.114 6 0.013 5.68 3 10 substrate for the enzyme. This is shown in Table I along with HI0398 22.6 6 1.1 9.2 6 0.4 0.063 6 0.014 1.46 3 10 the three other enzymes included in this study. For compari- Orf186 28.0 6 1.0 9.9 6 0.4 0.220 6 0.027 4.5 3 10 son, the two additional ADP-ribose pyrophosphatases de- 3 MJ1149 6.2 6 0.3 1.9 6 0.08 0.340 6 0.050 5.82 3 10 scribed in earlier works (7, 8) are also reported in the table. The activities toward ADP-ribose are compared with rates with some naturally occurring nucleoside diphosphate derivatives rates is in order. Kinetic parameters for ADP-ribose are com- known to be favored substrates for other members of the Nudix pared in Table II, and a broad distribution in some of the hydrolase family (8). In each case, ADP-ribose is the preferred constants is noted. These comparative values, derived under substrate, although there is a wide variation in absolute spec- standard assay conditions, should be interpreted with caution, ificities. For example, MJ1149 from the archaebacterium, because differences in the physiology and ecology of the indi- Methanococcus jannaschii, has no significant activity toward vidual entries could have large effects on the data. For exam- any of the other substrates, whereas Orf186 from E. coli and ple, we have shown that the V of MJ1149 increases 15-fold max H10398 from H. influenzae have substantial activities on when assayed at 75 °C (8), raising the rate from 6.2 to 93 NADH and GDP-mannose, respectively. However, more rigor- units/mg, and this temperature is still 10 °C below the normal ous kinetic analyses would be required for each of the putative habitat of the organism (9). substrates if a more substantive interpretation of the relative Other Properties of the Enzymes—As with most of the Nudix ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32321 FIG.3. Subfamilies of the Nudix hy- drolases. In each group, the amino acids constituting the Nudix box are in boldface type along with the downstream amino acid(s) distinguishing each subfamily. The check marks indicate proteins whose enzymatic activities have been positively identified. The source for Orf176 is J. D. Walsh and M. J. Bessman (unpublished results). hydrolases studied so far, the ADP-ribose pyrophosphatase Products of the Reaction—Aliquots of standard reaction mix- subfamily members have distinctly alkaline pH optima, rang- tures (omitting alkaline phosphatase), appropriately scaled up, ing from pH 8 to 9. All absolutely require a divalent cation for were analyzed as described under “Methods.” No inorganic activity, with Mg at approximately 2 mM the preferred metal. phosphate was formed during the course of the reactions. The Mn at equal concentrations is 10 –20% as effective. One of disappearance of substrate ADP-ribose was coincident with the the enzymes from this study, Orf209, has approximately 40% of appearance of AMP, and inorganic orthophosphate was re- 21 21 maximal activity when Mg is replaced by Zn , and this is leased upon incubation of the products with alkaline intestinal similar to the results seen previously for Orf186 (7). phosphatase. The course of the reaction may be written as 32322 ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases ADP-ribose 1 H O 3 AMP 1 ribose 5-phosphate. box, and in the latter, there is a consensus of eight amino acids in this region. When this 8-amino acid consensus sequence was Identification of Nudix Hydrolase Subfamilies—The discov- ery of a nucleoside triphosphatase activity associated with the first uncovered, there was only one known NADH pyrophos- E. coli mutT1 mutator gene (10) and also with its ortholog, phatase with the Nudix motif. We have recently cloned, ex- mutX,of S. pneumoniae (11, 12) suggested that the small pressed, purified, and determined the activities of the two region of amino acid identity in the two otherwise dissimilar additional entries XGLO67w (S. cerevisiae), and F13H10 (C. proteins comprised the catalytic site of these two enzymes. A elegans), both of which have the enzymatic activity predicted BLAST (2) search at that time revealed 13 other open reading from the downstream signals. The three-dimensional solution frames present in organisms ranging from viruses to humans structure for one of the Nudix hydrolases, MutT, has been (11, 13), and Koonin (13) suggested that the conserved MutT solved by NMR spectroscopy (17), and it has a unique, loop signature sequence might designate nucleoside triphosphate 1-helix-loop 2 motif encompassing its catalytic and nucleotide pyrophosphohydrolase activity. However, subsequent work has binding site. The characteristic amino acids distinguishing the revealed that the MutT enzyme is only one member of a large three families mentioned above would all be in loop 2 if this family of different enzymes with different substrates including structural feature were conserved in all of the Nudix hydro- sugar nucleotides, NADH, dinucleoside polyphosphates, and as lases. Preliminary studies on the crystal structure of the shown in the present work, ADP-ribose (for a review, see Ref. Orf209 ADP-ribose pyrophosphatase suggest that the loop- 8). Despite the large body of evidence to the contrary, the MutT helix-loop motif is present in this enzyme as well. The three- signature sequence (GX dimensional structures of two additional enzymes, Orf17, the EX REUXEEXGU) has been errone- 5 7 ously linked to the MutT enzymatic activity and to anti- dATPase (5), and Orf1.9, the GDP-mannose hydrolase (18), are mutagenesis. For example, MJ1149 of M. jannaschii has been also in the process of being solved, the former by x-ray crystal- 4 5 designated MutT in the TIGR sequencing project (14), whereas lography and the latter by NMR spectroscopy, so that we in reality it has neither nucleoside triphosphatase nor anti- should soon have insight into the generality of the loop-helix- mutagenic activity and is in fact a highly specific ADP-ribose loop motif in the Nudix hydrolase family. It is interesting to pyrophosphatase (8). Also, a recent report (15) attributes the note that one of the enzymes, Orf186, has both the conserved extreme radiation resistance of Deinococcus radiodurans to its proline and the conserved tyrosine (see Fig. 3, A and B). This large number of MutT genes insulating it from oxidative stress. correlates well with the specificity of Orf186, which is almost In fact, only one of the genes has been identified so far, gdr8, equally active on Ap A and ADP-ribose (Table I). On the other designating a new enzyme, UDP-glucose pyrophosphatase, hand, these amino acid predictors of activity should, at present, unrelated to the MutT enzyme. This ambiguity between the be viewed only as clues to narrow down the possibilities in “MutT motif,” an amino acid array shared by several different identifying new members of the family, since not all of the enzymes, and the “MutT enzyme,” connoting a specific physio- enzymes fit the present patterns. For example, two Ap A hy- logical function, has caused considerable confusion. For this drolases from the yeasts Schizosaccharomyces pombe (19) and reason, we introduced the acronym, Nudix hydrolase, to define S. cerevisiae (20) do not have the conserved tyrosine of the the family of different enzymes sharing the Nudix box signa- Ap A hydrolases, and Orf186, which also has substantial ac- ture sequence of amino acids, the MutT enzyme being only one tivity on NADH (see Table I) does not have the 8-amino acid member of this large family. Fig. 1 shows a Clustal (16) align- consensus predictive of this activity. ment of a partial list of putative enzymes containing the Nudix ADP-ribose Pyrophosphatase and Tellurite Resistance—An box (MutT motif) uncovered in a recent search of the data opportunity to test our classification scheme and to demon- banks using the BLAST program (2). Fig. 1 contains 70 entries strate its potential utility was provided by the trgB gene of R. selected from a total of 300 putative proteins from 75 different sphaeroides, which has been shown to be a tellurite resistance species. Sequestered in this list are different families of en- determinant (21). Fig. 3A, line 7, shows that TRGB contains zymes acting on the substrates mentioned above and almost the Nudix box and, in addition, the signal proline tentatively certainly some enzymes with novel, undiscovered activities. categorizing the protein as an ADP-ribose pyrophosphatase. To Since the Nudix box is common to all of these proteins, the test whether this enzyme could confer the tellurite resistance determinant(s) of specificity must be supplementary to the phenotype, we transformed E. coli with a plasmid, pTRC, con- Nudix signature sequence. One of our objectives in discovering taining the ADP-ribose pyrophosphatase gene (MJ1149) from and characterizing new members of this interesting family of the archaebacterium M. jannaschii, because the enzyme from enzymes is to recognize distinguishing features of the subfam- this organism is highly specific for ADP-ribose (see Table I). ilies in order to predict the enzymatic activity of undetermined The graphs in Fig. 4 clearly demonstrate that the ADP-ribose entries. As the collection of characterized enzymes grows, pyrophosphatase gene increases resistance to tellurite. In Fig. alignments of those with similar activities become more reveal- 4A, it can be seen that the transformed culture retained almost ing. Fig. 3A is an alignment of the ADP-ribose pyrophosphatase 100% viability at a 50% survival rate for the parent culture. subfamily showing a highly conserved proline, 15 or 16 amino Likewise, there was approximately 60% survival versus 100% acids downstream of the terminal glycine of the Nudix box. The killing, at 0.5 mg/ml K TeO . Fig. 4B shows the differential 2 3 checked entries have all been identified, and the remaining effect of tellurite on growing cultures of the transformed and entries are predicted. Actually, we predicted the activity of the parent cells. That this is a specific effect of the ADP-ribose H. influenza and B. subtilis proteins before their respective pyrophosphatase gene and not a general property of the Nudix genes were cloned and expressed, and we have recently learned hydrolases is shown in Fig. 4C. None of the other genes tested that a human EST (Fig. 3A, line 17) is also an ADP-ribose confer tellurite resistance. Commensurate with the increase in pyrophosphatase Similar alignments are shown in Fig. 3, B tellurite resistance was a 5-fold increase in ADP-ribose pyro- and C, for diadenosine polyphosphate hydrolases and the phosphatase in crude extracts of the transformed cells com- NADH hydrolases, respectively. In the former, there is a con- pared with the parent culture. served tyrosine 16 –18 amino acids downstream from the Nudix W-L. Xu and M. J. Bessman, unpublished results. S. Gabelli, M. J. Bessman, and L. M. Amzel, unpublished results. 1 5 C. A. Dunn, S. Desai, and M. J. Bessman, unpublished results. P. Leggett, M. J. Bessman, and A. S. Mildvan, unpublished results. 2 6 H. Yang, M. Slupska, and J. H. Miller, personal communication. C. A. Dunn and M. J. Bessman, unpublished observations. ADP-ribose Pyrophosphatase Subfamily of Nudix Hydrolases 32323 Tellurite resistance has been used for many years in the differential diagnosis of pathological microorganisms and is a complex process implicating many genes found in prokaryotes and eukaryotes (for a review, see Ref. 22). Our experiments and classification scheme strongly suggest that one of these genes, trgB, designates an ADP-ribose pyrophosphatase, implicating ADP-ribose itself as a factor in tellurite sensitivity. Although the role played by ADP-ribose in this process is not known, these experiments are an example of how functional genomics, the prediction of protein function from amino acid sequence, can aid in identifying the activities of unknown proteins in- volved in physiological processes. It is, perhaps, not surprising that ADP-ribose is directly involved in, or potentiates, tellurite toxicity. Cellular ADP- ribose arises from the hydrolysis of mono- and poly(A)DP- ribosylated proteins during the regulation of metabolic pro- cesses (for a review, see Ref. 23) and also by the large scale turnover of NAD , amounting to approximately 30 and 90% of the total NAD synthesis in E. coli and HeLa cells, respectively (24). Because of its free aldehydic group, ADP-ribose can deri- vatize terminal amino groups, lysines, and cysteines in pro- teins nonenzymatically (25, 26), thereby inactivating enzymes or leading to proteins targeted for apoptosis (27–29) or to non- specifically tagged proteins confusing the ADP-ribosylation recognition system. Recently, the accumulation of ADP-ribose has been implicated in the liver damage caused by high levels of acetaminophen (N-acetyl-p-aminophenol, Tylenol ) when it was shown that the acetaminophen metabolite, N-acetyl-p- benzoquinonimine, inhibits rat liver ADP-ribose pyrophos- phatase (30). We have also seen that this derivative inhibits YSA1, the yeast enzyme described in this paper. It has also been reported that inhibitors of poly(ADP-ribose) polymerase, one of the major sources of cellular ADP-ribose, prevent the liver damage caused by overdoses of acetaminophen (31). These recent experiments support the large body of data implicating free ADP-ribose as a cytotoxic agent. As mentioned in the Introduction, we have suggested that the members of the Nudix hydrolase family of enzymes share common features including a conserved amino acid signature sequence and a specificity for nucleoside diphosphate deriva- tives and that one of their physiological functions is to sanitize the cell of potentially toxic metabolites. The ubiquitous ADP- ribose pyrophosphatases described in this paper meet these three criteria and qualify this subfamily as a bona fide member of the Nudix hydrolases. Acknowledgments—We are indebted to Drs. Samuel Kaplan and Mark Gomelsky for help with trgB. 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