Orf186 Represents a New Member of the Nudix Hydrolases, Active on Adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH*

Suzanne F. O'Handley, David N. FrickDagger , Christopher A. Dunn, and Maurice J. Bessman§

From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

orf186, a new member of the Nudix hydrolase family of genes, has been cloned and expressed, and the protein has been purified and identified as an enzyme highly specific for compounds of ADP. Its three major substrates are adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH, all implicated in a variety of cellular regulatory processes, supporting the notion that the function of the Nudix hydrolases is to monitor the concentrations of reactive nucleoside diphosphate derivatives and to help modulate their accumulation during cellular metabolism.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The Nudix hydrolases are a family of widely distributed enzymes (1) characterized by a structural motif, the Nudix box, having the highly conserved consensus sequence
<UP><B>G</B></UP><IT>XXXXX</IT><UP><B>E</B></UP><IT>XXXXXXX</IT><UP><B>REU</B></UP><IT>X</IT><UP><B>EE</B></UP><IT>X</IT><UP><B>GU</B></UP>
<UP><SC>Sequence 1</SC></UP>
where U represents one of the bulky hydrophobic amino acids, usually I, L, or V. Based on predictive (2) and structural (3) studies, the Nudix box designates a unique loop-helix-loop motif, involved in the binding of the substrate (4) and in catalysis (5, 6). Searches of the protein data banks have uncovered over 100 putative proteins containing the Nudix structural motif, within all three kingdoms, the prokaryota, archaea, and eukaryota, in organisms ranging in complexity from viruses to humans. To date, about 20 of these proteins have been identified as enzymes, and their substrates include nucleoside triphosphates, co-enzymes, sugar nucleotides, and dinucleoside polyphosphates. Although seemingly diverse, these compounds share a common structural feature. They are all nucleoside diphosphates linked to some other moiety, x; hence the acronym, Nudix (1). In addition to the structural similarity of these substrates, they are all either potentially toxic or deleterious compounds, cell signaling molecules, regulators, or metabolic intermediates whose concentrations require modulation during the vicissitudes of the cellular environment.

The subject of this paper, Orf186, was identified in a BLAST search (7) as a putative 186-amino acid protein, locus YRFE, in Escherichia coli (GenBank accession no. P45799). Herein, we describe the cloning and expression of the orf186 gene, and the purification and initial characterization of the resulting protein. Orf186 is a novel enzyme, contains the Nudix box, and hydrolyzes the sensitive metabolic intermediates, adenosine(5')triphospho(5')adenosine (Ap3A),1 ADP-ribose, and NADH, all derivatives of the nucleoside diphosphate, ADP. Thus, Orf186 is a new member of the Nudix hydrolase family.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Primers were obtained from Integrated DNA Technologies (Coralville, IA). Biochemicals and enzymes were purchased from Sigma unless otherwise noted. E. coli strain MG1655 was kindly provided by Dr. Frederick R. Blattner (University of Wisconsin). Other common organisms and vectors were from laboratory stocks.

Methods

Cloning-- The orf186 gene was amplified from E. coli strain MG1655 chromosomal DNA using the polymerase chain reaction. The oligonucleotides d(GCGCGCGGTACCGATGAGCAAATCATTACAAAAACC) and d(GCGCGCGGATCCTTACACTCGCCCCTGCCC) were used as primers to amplify the gene and incorporate a KpnI restriction site at the start of the gene and a BamHI restriction site at the end of the gene. The amplified gene was purified by agarose gel electrophoresis, digested with KpnI and BamHI, and ligated into the KpnI and BamHI restriction sites of plasmid pTRC99A. The resulting plasmid pTRCorf186 was used to transform E. coli strain HB101.

The orf186 gene was subcloned from pTRCorf186 using a modification (8) of the megaprimer method (9) for site-directed mutagenesis. The oligonucleotides d(GGCCGCTGGCGCACATGATGGATTTG) and d(GCCAGGCAAATTCTG) were used to create a megaprimer containing a silent mutation of T to C at position 462 of orf186, thereby eliminating the NdeI site in the orf186 gene but retaining the histidine encoded for at this position. The purified megaprimer and the oligonucleotide d(GCGCGCGCATATGAGCAAATCATTACAAAAACC) were used as primers to amplify the orf186 gene, incorporating the mutation described above, as well as an NdeI site at the start of the gene, and a BamHI site at its end. The amplified gene was digested with NdeI and BamHI restriction enzymes and ligated into the NdeI and BamHI restriction sites of plasmid pET11b to place the orf186 gene under control of a T7 lac promoter for expression. The resulting plasmid, pETorf186(mut462) was used to transform E. coli strain DH5alpha for storage and E. coli strain BL21(DE3) for expression.

Purification of the Enzyme-- E. coli strain BL21(DE3) containing pETorf186(mut462) was grown at 37 °C in three stages. A single colony was inoculated into 10 ml of LB medium containing 100 µg/ml ampicillin and grown to an A600 of 0.4. The cells were collected by centrifugation, washed with 0.9% NaCl, and added to 100 ml of fresh LB medium plus ampicillin. These cells were grown to an A600 of 0.6, collected and washed as above, and added to 2 liters of fresh medium plus ampicillin. When growth reached an A600 of 0.6, the culture was induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside and grown for an additional 2 h.

The induced cells were harvested, washed by suspension in a buffered saline solution, resuspended in 2 volumes of buffer A (50 mM Tris, pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol), sonicated, centrifuged, and the precipitate was discarded. The protein concentration of the supernatant was adjusted to 10 mg/ml with buffer A (Fraction I). A typical preparation yielded approximately 200 mg of protein from 3 g of cells.

To Fraction I was added 0.1 volume of 10% streptomycin sulfate, and after 15 min on ice, the precipitate was discarded. Approximately 60% of the enzymatic activity remained in the supernatant. The concentration of streptomycin was raised to 2% with 25% streptomycin sulfate, and this solution was stored on ice overnight. The precipitate was collected and dissolved in 4 ml of buffer A containing 10% ammonium sulfate (Fraction II). Fraction II was loaded onto a 2.5 × 60-cm column of Sephadex G100 and eluted with buffer A containing 50 mM NaCl. The fractions containing the bulk of the enzyme were combined (Fraction III), and concentrated by precipitation in 45% ammonium sulfate (Fraction IV). The purified enzyme was stable for months when stored at -80 °C.

Enzyme Assay-- In the standard assay, the hydrolysis of Ap3A to AMP and ADP was measured by converting the products to Pi and adenosine with calf intestinal phosphatase. The reaction mixture contained (in 50 µl): 2 mM Ap3A, 50 mM Tris, pH 9, 0.5 mM MgCl2, 1% glycerol, 0.1 mg/ml bovine serum albumin, 0.2 unit of calf intestinal phosphatase, and 0-1 milliunit of enzyme. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 250 µl of 4 mM EDTA. The Pi produced was measured by the colorimetric method of Ames and Dubin (10). A unit of Orf186 cleaves 1 µmol of substrate/min.

The identity of the products and the stoichiometry of the reaction were analyzed by paper electrophoresis (11) and high performance liquid chromatography in scaled-up standard reaction mixtures, from which calf intestinal phosphatase was omitted.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Subcloning, Expression, and Purification

Elimination of the NdeI restriction site in the wild type orf186 gene, by making a silent mutation at position 462, enabled us to place orf186 between the NdeI and BamHI sites of pET11b, directly down stream from a ribosome binding site and under control of a T7 lac promoter for optimal expression. The sequence of the cloned gene agrees with that reported for orf186 (accession no. P45799), except for the engineered T right-arrow C transition at position 462 (data not shown).

The pETorf186 plasmid was unstable in E. coli strain BL21(DE3) when the culture was grown to high cell density, presumably due to a selective disadvantage upon depletion of ampicillin. Therefore, the cultures were grown stepwise, centrifuged, and suspended in fresh medium containing ampicillin, as described under "Experimental Procedures." With this procedure, virtually 100% of the cells retained the plasmid, as determined by plating aliquots on selective agar.

Expression of the gene results in the appearance of a major band on a denaturing gel (Fig. 1), corresponding to a 21.5-kDa protein not detectable in the strain containing pET11b without the insert. When the crude extract is brought to a concentration of 1% streptomycin sulfate to remove nucleic acids, approximately 40% of the enzyme precipitates and 60% remains in solution. Increasing the concentration of streptomycin in the supernatant to 2% results in a quantitative precipitation of the remainder of the enzyme, while leaving many of the other proteins behind. Subsequent fractionation by gel filtration and concentration by ammonium sulfate precipitation led to a preparation substantially free of contaminating proteins (Fig. 1). Orf186 migrated on the SDS gel as expected for the 21-kDa polypeptide predicted from its amino acid composition. However, when chromatographed on a pre-calibrated gel filtration column, it appeared as a symmetrical peak in a region expected for a 43-kDa protein. Thus, it is quite possible that the enzyme exists as a homodimer in its native state; however, at present, no further work has been done to investigate this issue.


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Fig. 1.   Expression and purification of the Orf186 protein. A polyacrylamide gel (15%) containing 1% SDS, stained with Coomassie Blue, contained the following: lane 1, reference proteins including serum albumin (66 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and alpha -lactalbumin (14 kDa); lanes 2 and 3, 1 µl of crude extracts from cells transformed with pET11b (no insert) or pETorf186, respectively; lane 4, enzyme Fraction IV, containing approximately the same number of enzyme units as present in lane 3.

Properties of the Enzyme

Requirements of the Reaction-- The enzyme has optimal activity at a distinctly alkaline pH. In Tris buffer, the maximum rate of hydrolysis for Ap3A is at pH 9.0, with the rate falling to <50% at pH 7.5. In glycine buffer, the maximum is at pH 10 with a 50% reduction in rate at pH 8. This alkaline pH optimum is typical of four other Nudix hydrolases we have characterized, namely MutT dGTPase, pH 9 (12); NADH pyrophosphatase, pH 8.5 (13); GDP-mannose hydrolase, pH 9.3 (14); and Orf17 dATPase, pH 8.6 (15). These relatively high pH optima may implicate the guanido group of arginine in the binding of the pyrophosphate-containing substrates, and in the mechanism of catalysis, as has been implied for several other enzymatic reactions involving phosphate esters (16). We have noted that arginine is one of the four absolutely conserved amino acids in all of the proteins containing the Nudix box (1), and we have observed that the Nudix hydrolases are inactivated by carbonyl reagents targeting arginine.2

The enzyme has an absolute requirement for a divalent cation, achieving optimal activity with 0.5 mM Mg2+, but Zn2+ and Mn2+ can partially substitute, sustaining rates of hydrolysis of 34% and 17%, respectively, on Ap3A. No activity was detected in the presence of Co2+ or Ca2+. These metal ion effects clearly distinguish the Orf186 enzyme from another Ap3A hydrolase purified from E. coli (17) that cannot substitute Mg2+ with Mn2+ or Zn2+. Furthermore, it does not hydrolyze NAD+ as does Orf186 (see below), and it has a specific activity (units per mg) about 300-fold lower than Orf186.

Specificity-- Table I lists a number of biochemicals tested as potential substrates for Orf186. Several features are noteworthy. In contrast to MutT, the first member of the Nudix family studied (18), and Orf17 (15), both of which hydrolyze ribo- and deoxyribonucleoside triphosphates, Orf186 has no detectable activity on these compounds. Instead, it seems to be specific for nucleoside pyrophosphates in which one of the moieties is ADP. Thus, Ap2A, Ap3A, ADP-ribose, and NADH are all good substrates, as well as Ap3G and Ap37-methyl-G. However, when the remaining adenine is replaced by guanine, as in Gp3G, the activity falls dramatically. Activity on the sugar nucleotides reinforces this observation. Both ADP-glucose and ADP-mannose, although not indigenous to E. coli, are hydrolyzed at a significant rate, whereas the corresponding GDP- and UDP-sugars, more characteristic metabolites, are not. Additionally, when adenine is replaced by hypoxanthine as in deamino-NADH, the presence of the 6-amino instead of a keto group reduces the activity 100 fold.

                              
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Table I
Substrate specificity of Orf186
All substrates were present at a concentration of 2 mM and were assayed using the colorimetric procedure described under "Experimental Procedures" except for the (deoxy)nucleoside triphosphates, which were assayed as described in Ref. 15.

In the ApnA series, activity falls off sharply when n > 3. This could be due to steric constraints at the active site of the enzyme, or to electrostatic effects related to the additional phosphates, since ATP, itself, is not a significant substrate. Table II compares kinetic constants for some of the favored substrates. Their turnover numbers (kcat) are all in the same range. This is true, also, for the Km values of the first four compounds. However, the Km for NAD+ is approximately 100-fold higher than the rest, leading to a "catalytic efficiency" (kcat/Km) 50-80-fold lower than the other substrates. This may have important physiological consequences for the cell (see "Discussion").

                              
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Table II
Kinetic parameters for Orf186
The standard assay as described under "Experimental Procedures" was used with concentrations of 0.05-2 mM for all substrates except NAD+ which was present at concentrations of 0.5-50 mM. Km and Vmax were determined from a non-linear regression analysis (53), and kcat was calculated from Vmax assuming one active site per monomer. A unit of enzyme hydrolyzes 1 µmol of substrate/min.

Products of the Reaction-- The course of hydrolysis of Ap3A was measured in a standard reaction mixture (minus alkaline phosphatase) scaled up 20-fold. At intervals, aliquots were removed and analyzed by high performance liquid chromatography. Fig. 2 shows the disappearance of Ap3A and the commensurate appearance of AMP and ADP. For every molecule of Ap3A hydrolyzed, one molecule each of AMP and ADP was formed, e.g. at 7.5 min, 37 nmol of Ap3A were lost, and 35 and 34 nmol of AMP and ADP were formed, respectively. At 30 min, the values (in order) were 69, 67, and 67. No Pi was detected throughout the course of the reaction. Therefore, the reaction may be written as shown below.
<UP>Ap</UP><SUB>3</SUB><UP>A</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP>→<UP>AMP</UP>+<UP>ADP</UP>
<UP><SC>Reaction</SC></UP> 1
The products formed from NADH, ADP-ribose, Ap2A, Ap3G, and adenosine(5')tetraphospho(5')adenosine (Ap4A) were also examined. In each case, AMP was always formed as one of the products, and the remainder of the molecule was liberated intact. Two of the substrates are of special interest.
<UP>Ap</UP><SUB>3</SUB><UP>G</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP>→<UP>AMP</UP>+<UP>GDP</UP>
<UP><SC>Reaction</SC></UP> 2
Neither GMP nor ADP is produced, indicating that the substrate is oriented asymmetrically at the catalytic site. Nucleophilic attack by water could occur on the alpha  or beta  phosphorus (with respect to the adenosine moiety) leading to the observed products. It is noteworthy that H218O studies have shown that two other Nudix hydrolases, the MutT dGTPase and the Orf17 dATPase, attack the less favorable (more shielded) beta  phosphorus, forming dGMP (or dAMP) and PPi (15, 19). It would be of interest to do similar studies with the Orf186 diadenosine triphosphatase to test the generality of this mechanism. Another substrate worth noting is Ap4A. Although it is hydrolyzed at only 1% of the rate of Ap3A, our analysis of the products (data not shown) indicate that it is cleaved according to the following reaction.
<UP>Ap</UP><SUB>4</SUB><UP>A</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP>→<UP>AMP</UP>+<UP>ATP</UP>
<UP><SC>Reaction</SC></UP> 3
This is the first example of an "asymmetric" Ap4A hydrolase in prokaryotes. Previous reports had relegated this activity to eukaryotes, since all other enzymes prepared from eubacteria or archaea hydrolyze Ap4A "symmetrically," producing 2 mol of ADP (for review, see Ref. 20).


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Fig. 2.   Products of the reaction. The standard enzyme assay as described under "Experimental Procedures" was scaled up 20-fold and monitored by high performance liquid chromatography to quantify the substrate, Ap3A (black-triangle) and the products, AMP (open circle ) and ADP (bullet ).

    DISCUSSION
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Introduction
Procedures
Results
Discussion
References

Fig. 3 shows the results of a recent BLAST search (7) for polypeptide sequences homologous to the Nudix box. The broad biodiversity is self-evident, suggesting that representatives of this family of proteins are of primordial origin and were conserved during evolution. We have instituted a systematic study of the members of the group to delimit the scope of reactions they catalyze, and to ascertain whether they exhibit a functional commonality in cellular metabolism. At present, the genes coding for 11 of the proteins listed in Fig. 3 have been cloned, and the expressed proteins were identified as enzymes. These are shown in Fig. 4 along with their major substrates, which, although quite different in structure and metabolic origin, are all derivatives of nucleoside diphosphates. The first of these enzymes, the MutT nucleoside triphosphatase (12, 18), was found to be defective in the original mutT1 mutator strain (21), thereby accounting for the >1000-fold increase in spontaneous mutation frequency in organisms defective in this gene (22). Since then, homologous enzymes containing the Nudix box have been found in Streptococcus pneumoniae (6), Proteus vulgaris (23), human (24), rat (25), and mouse (26). This subset of Nudix hydrolases most likely prevent the specific AT right-arrow CG transversions seen in the mutator strains (27) by sanitizing the nucleotide pool of a mutagenic form of dGTP (12), possibly 8-oxo-dGTP (28). The next enzyme in Fig. 4, Orf17 dATPase (15), may play a similar role in hydrolyzing the recently discovered, and potentially mutagenic, 2-hydroxy-dATP (29), or in monitoring the accumulation of dATP, the major negative effector of deoxynucleotide synthesis (30). The following enzyme, Orf257, is an unusual NADH pyrophosphatase, 100 times more active on the reduced form of the coenzyme than on NAD+ (13). It could play a role in maintaining the pivotal cellular NADH/NAD+ ratio important in balancing the anabolic versus catabolic pathways, as has been suggested previously for hydrolases active on NAD+ (31). The next enzyme, GDP-mannose hydrolase, could play a role in recycling nucleoside diphosphate sugar intermediates and diverting them for reutilization in different pathways during cell maturation, as originally suggested for other sugar nucleotides (32, 33). Also in Fig. 4, the Ap4A hydrolase, purified from human placenta (34) contains the Nudix box signature sequence, as does the enzyme from pig (35, 36). Ap4A is a member of the general class of diadenosine polyphosphates, ApnA, where n = 2-6. Since the discovery of these compounds (37), considerable interest has been generated by reports of their involvement in several areas of cell physiology including the responses to heat shock, oxidative stress, and starvation, in cell proliferation and DNA replication and repair, as neurotransmitters, effectors of platelet aggregation, vasotone regulators etc. (for reviews, see Refs. 38 and 39). Although the mechanism(s) of action of these compounds is still in question, the plethora of reports citing their complicity augurs their importance.


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Fig. 3.   Members of the Nudix family. Polypeptides identified in a Blast search (7) against the 23 amino acids spanning the Orf186 signature sequence are shown.


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Fig. 4.   Enzymes of the Nudix hydrolase family. Listed are those proteins containing the Nudix box characterized as enzymes, along with their preferred substrates.

Orf186, described in this paper, is a bona fide member of the Nudix hydrolases, of particular interest, since it recognizes three different substrates characteristic of this family of enzymes. A role played by its NADH hydrolase activity in maintaining the optimal NADH/NAD+ ratio, referred to above, may be of particular importance during periods of anaerobiosis, since the essentially reversible NAD+-linked oxidoreduction reactions would shut down due to the accumulation of NADH in the absence of acceptor oxygen.

A second major substrate of Orf186 is Ap3A, a member of the broadly distributed ApnA family implicated in the diverse physiological responses mentioned above. Recently, Barnes et al. (40) reported that the human fragile histidine triad protein (FHIT) is an Ap3A hydrolase. This enzyme is the product of the putative human tumor suppressor gene, FHIT, at 3p14.2, found to be aberrant in cell lines derived from cancers of the lung, esophagus, stomach, and colon (41, 42). The connection between neoplasia and a defect in Ap3A hydrolysis is not readily apparent, but it focuses attention on this member of the ApnA family and stimulates interest in the role of Ap3A in E. coli metabolism, which may be more amenable to investigation than human cell lines.

The third significant substrate, ADP-ribose, is a by-product of the ubiquitous NAD+-linked ADP-ribosylation reactions implicated in a broad range of cellular regulatory processes (for review, see Refs. 43 and 44). Hydrolytic removal of the mono- or poly(ADP-ribosyl) groups from modified proteins produces free ADP-ribose. In addition, the normal cellular turnover of NAD+ itself, which amounts to approximately 30% and 90% of the total NAD+ synthesis in E. coli and HeLa cells, respectively (45), is in part due to the production of ADP-ribose and nicotinamide by NAD+-glycohydrolase. A role for Orf186 would be the recycling of the ADP-ribose moiety thus formed. Perhaps a more important function is to sanitize the metabolic pool of the potentially harmful accumulation of ADP-ribose. Because of its free aldehydic group, ADP-ribose can modify proteins in non-enzymatic glycation reactions, derivatizing terminal amino groups, cysteines, and lysines (46, 47). Non-enzymatic glycation of long-lived proteins by reducing sugars increases with age, producing what have been called advanced glycosylation end products, which may be targets for degradation and thereby trigger apoptosis in eukaryotic cells (48-50). Ridding the metabolic pool of ADP-ribose may be especially important for two reasons. First, ADP-ribose is a more active glycating agent, by several orders of magnitude, than the free sugars (51). Second, random or nonspecific ADP-ribosylation could misdirect the cellular recognition systems to, or away from, impostors masquerading as properly tagged proteins. A testimony to the importance of this ADP-ribose pyrophosphatase activity is its widespread distribution. We are presently characterizing an additional highly active ADP-ribose hydrolase from E. coli (Orf209), and other ADP-ribose pyrophosphatases from yeast (YSA1), from Bacillus subtilis (YQKG), from Haemophilus influenzae (YZZG), and from an archaeon, Methanococcus janaschii (MJ1149), all of which contain the Nudix box.3 One of the human genome expressed sequence tags (EST 348875) codes for a Nudix polypeptide highly homologous to the yeast (YSA1) enzyme, and specific ADP-ribose pyrophosphatases have been partially purified from rat liver and Artemia (52) although the sequences of the latter two enzymes have not been reported.

At present, no mutants defective in Orf186 are available to test whether the loss of these enzymatic activities elicits a recognizable phenotype. We are in the process of constructing a null mutant of the yeast YSA1 gene (mentioned above), coding for a highly active ADP-ribose pyrophosphatase, to address the question of phenotype. However, ablating one gene may not seriously compromise the cell, since in our experience, there are several Nudix hydrolases with overlapping specificities that might compensate for the loss of one. This is not altogether unexpected, if the enzymatic process is important for survival. In this respect, it is interesting to note that in a recent search of the partially sequenced genome of Deinococcus radiodurans, an organism noted for its extraordinary resistance to ionizing radiation, we have identified 17 distinct polypeptides containing the Nudix box.

Orf186 has proven to be an interesting new member of the Nudix hydrolases, a broadly distributed family of enzymes sharing a common structural motif, a related series of substrates and, perhaps, a common function, i.e. securing the cellular environment. As a result of the vigorous activity in cloning and sequencing of the various genomes, new candidates containing the Nudix box motif are being uncovered at a rapid pace. At present, over 100 have been reported in various prokaryotes, 5 in yeast, and in terms of expressed sequence tags, 2 in rice, 3 in Caenorhabditis elegans, 2 in Arabidopsis, 4 in mouse, and 7 in human. In our limited experience, the Nudix hydrolase activities so far identified in the prokaryotes and archaea have their counterparts in yeast and and higher eukaryotes. If this pattern continues, the use of prokaryotes to discover and characterize new members of this family should simplify the task of identifying their activities, and perhaps their physiological functions in the more complex organisms.

    ACKNOWLEDGEMENTS

We thank Dr. Frederick R. Blattner for E. coli strain M1655 and The Institute for Genomic Research for making sequence data available prior to publication.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM18649. This is Publication 1516 from the McCollum-Pratt Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Biological Chemistry, Harvard Medical School, Boston, MA 02115.

§ To whom correspondence should be addressed. Tel.: 410-516-7316; Fax: 410-516-5213; E-mail: zoot{at}jhu.edu.

1 The abbreviations used are: Ap3A, adenosine(5')triphospho (5')adenosine; Ap2A, adenosine(5')diphospho(5')adenosine; Ap4A, adenosine(5')tetraphospho(5')adenosine.

2 C. A. Dunn and M. J. Bessman, unpublished observations.

3 C. A. Dunn, S. F. O'Handley, S. Sheikh, D. N. Frick, K. Finney, and M. J. Bessman, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Bessman, M. J., Frick, D. N., and O'Handley, S. F. (1996) J. Biol. Chem. 271, 25059-25062[Free Full Text]
  2. Koonin, E. V. (1993) Nucleic Acids Res. 21, 4847[Medline] [Order article via Infotrieve]
  3. Abeygunawardana, C., Weber, D. J., Gittis, A. G., Frick, D. N., Lin, J., Miller, A.-F., Bessman, M. J., Mildvan, A. S. (1995) Biochemistry 34, 14997-15005[Medline] [Order article via Infotrieve]
  4. Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., Mildvan, A. S. (1997) Biochemistry 36, 1199-1211[CrossRef][Medline] [Order article via Infotrieve]
  5. Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., Mildvan, A. S. (1996) Biochemistry 35, 6715-6726[CrossRef][Medline] [Order article via Infotrieve]
  6. Mejean, V., Salles, C., Bullions, L. C., Bessman, M. J. (1993) Mol. Microbiol. 11, 323-330
  7. Altschul, S. F., Gish, W., Meyers, E. W., Lipman, D. J. (1990) J. Mol. Biol. 203, 403-410[CrossRef]
  8. Barik, S. (1993) in PCR Protocols: Current Methods and Applications (White, B. A., ed), Vol. 15, pp. 277-286, Humana Press Inc., Totowa, NJ
  9. Sarkar, G., and Sommer, S. S. (1990) BioTechniques 8, 404-407 [Medline] [Order article via Infotrieve]
  10. Ames, B. N., and Dubin, D. T. (1960) J. Biol. Chem. 235, 769-775[Medline] [Order article via Infotrieve]
  11. Markham, R., and Smith, J. D. (1952) Biochem. J. 52, 552-557
  12. Bhatnagar, S. K., Bullions, L. C., and Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054[Abstract/Free Full Text]
  13. Frick, D. N., and Bessman, M. J. (1995) J. Biol. Chem. 270, 1529-1534[Abstract/Free Full Text]
  14. Frick, D. N., Townsend, B. D., and Bessman, M. J. (1995) J. Biol. Chem. 270, 24086-24091[Abstract/Free Full Text]
  15. O'Handley, S. F., Frick, D. N., Bullions, L. C., Mildvan, A. S., Bessman, M. J. (1996) J. Biol. Chem. 271, 24649-24654[Abstract/Free Full Text]
  16. Riordan, J. F., McElvaney, K. D., and Borders, C. L. J. (1977) Science 195, 884-886[Medline] [Order article via Infotrieve]
  17. Hurtado, C., Ruiz, A., Sillero, A., and Sillero, M. A. G. (1987) J. Bacteriol. 169, 1718-1723[Medline] [Order article via Infotrieve]
  18. Bhatnagar, S. K., and Bessman, M. J. (1988) J. Biol. Chem. 263, 8953-8957[Abstract/Free Full Text]
  19. Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942[Abstract/Free Full Text]
  20. Guranowski, A., and Sillero, A. (1992) in Ap4A and Other Dinucleoside Polyphosphates (McClennan, A. G., ed), pp. 81-133, CRC Press, Inc, Boca Raton
  21. Bhatnagar, S. K., Bullions, L. C., Lew, G., and Bessman, M. J. (1990) J. Bacteriol. 172, 2802-2803[Medline] [Order article via Infotrieve]
  22. Treffers, H. P., Spinelli, V., and Belser, N. O. (1954) Proc. Natl. Acad. Sci. U. S. A. 40, 1064-1071
  23. Kamath, A. V., and Yanofsky, C. (1993) Gene (Amst.) 134, 99-102[CrossRef][Medline] [Order article via Infotrieve]
  24. Sakumi, K., Furuichi, M., Tsuzuki, T., Kakuma, T., Kawabata, S., Maki, H., and Sekiguchi, M. (1993) J. Biol. Chem. 268, 23524-23530[Abstract/Free Full Text]
  25. Cai, J. P., Kakuma, T., Tsuzuki, T., and Sekiguchi, T. (1995) Carcinogenesis 16, 2343-2350[Abstract]
  26. Kakuma, T., Nishida, J., Tsuzuki, T., and Sekiguchi, M. (1995) J. Biol. Chem. 270, 25942-25948[Abstract/Free Full Text]
  27. Yanofsky, C., Cox, E., C., and Horn, V. (1966) Genetics 55, 274-281
  28. Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275[CrossRef][Medline] [Order article via Infotrieve]
  29. Kamiya, H., and Kasai, H. (1995) J. Biol. Chem. 270, 19446-19450[Abstract/Free Full Text]
  30. Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 48, 133-158[CrossRef][Medline] [Order article via Infotrieve]
  31. McCreanor, G., and Bender, D. (1983) Biochim. Biophys. Acta 759, 222-228[Medline] [Order article via Infotrieve]
  32. Glaser, L. (1965) Biochim. Biophys. Acta 101, 6-15[Medline] [Order article via Infotrieve]
  33. Sonnino, S., Carminatti, H., and Cabib, E. (1966) Archiv. Biochem. Biophys. 116, 26-33[Medline] [Order article via Infotrieve]
  34. Lazewska, D., Starzynska, E., and Guranowski, A. (1993) Protein Exp. Purif. 4, 45-51[CrossRef][Medline] [Order article via Infotrieve]
  35. Thorne, N. M. H., Hankin, S., Wilkinson, M. C., Nunez, C., Barraclough, R., McLennan, A. G. (1995) Biochem. J. 311, 717-721[Medline] [Order article via Infotrieve]
  36. Hankin, S., Wintero, A. K., and McLennan, A. G. (1997) Int. J. Biochem. Cell Biol. 29, 317-323[CrossRef][Medline] [Order article via Infotrieve]
  37. Zamecnik, P. C., Stephenson, M. L., Janeway, C. M., Randerath, K. (1966) Biochem. Biophys. Res. Commun. 24, 91-97[Medline] [Order article via Infotrieve]
  38. Baxi, M. D., and Vishwanatha, J. K. (1995) J. Pharmacol. Toxicol. Methods 33, 121-128[CrossRef][Medline] [Order article via Infotrieve]
  39. McLennan, A. G. (ed) (1992) Ap4A and Other Dinucleoside Polyphosphates, CRC Press, Inc., Boca Raton, FL
  40. Barnes, L. D., Garrison, P. N., Siprashvili, Z., Guranowski, A., Robinson, A. K., Ingram, S. W., Croce, C. M., Masataka, O., Huebner, K. (1996) Biochemistry 35, 11529-11535[CrossRef][Medline] [Order article via Infotrieve]
  41. Ohta, M., Inoue, H., Cotticelli, M. G., Kastury, K., Baffa, R., Palazzo, J., Siprashvili, Z., Mori, M., McCue, P, Druck, T., Croce, C. M., Huebner, K. (1996) Cell 84, 587-597[Medline] [Order article via Infotrieve]
  42. Sozzi, G., Veronese, M. L., Negrini, M., Baffa, R., Cotticelli, M. G., Inoue, H., Tornielli, S., Pilotti, S., De Gregorio, L., Pastorino, U., Pierotti, M. A., Ohta, M., Huebner, K., Croce, C. M. (1996) Cell 85, 17-26[Medline] [Order article via Infotrieve]
  43. Ueda, K., and Hayaishi, O. (1985) Annu. Rev. Biochem. 54, 73-100[CrossRef][Medline] [Order article via Infotrieve]
  44. Moss, J., and Zahradka, P. (eds) (1994) Mol. Cell. Biochem. 138
  45. Olivera, B. M., Hughes, K. T., Cordray, P., and Roth, J. R. (1989) in ADP-Ribose Transfer Reactions (Jacobson, M. K., and Jacobson, E. L., eds), pp. 353-360, Springer-Verlag, New York
  46. Jacobson, E. L., Cervantes-Laurean, D., and Jacobson, M. K. (1994) Mol. Cell. Biochem. 138, 207-212[Medline] [Order article via Infotrieve]
  47. McDonald, L. J., and Moss, J. (1994) Mol. Cell. Biochem. 138, 221-226[Medline] [Order article via Infotrieve]
  48. Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5588-5592[Abstract]
  49. Bucala, R., and Cerami, A. (1992) Adv. Pharmacol. 23, 1-34[Medline] [Order article via Infotrieve]
  50. Kaneto, H., Fujii, J., Myint, T., Miyazawa, N., Islam, K. N., Yamasaki, Y., Taniguchi, M. (1996) Biochem. J. 320, 855-863[Medline] [Order article via Infotrieve]
  51. Cervantes-Laurean, D., Jacobson, E. L., Jacobson, M. K. (1996) J. Biol. Chem. 271, 10461-10469[Abstract/Free Full Text]
  52. Fernandez, A., Ribeiro, J. M., Costas, M. J., Pinto, R. M., Canales, J., Cameselle, J. C. (1996) Biochim. Biophys. Acta 1290, 121-127[Medline] [Order article via Infotrieve]
  53. Cleland, W. W. (1979) Methods Enzymol. 63, 103-139[Medline] [Order article via Infotrieve]


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