From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218
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ABSTRACT |
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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.
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INTRODUCTION |
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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
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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.
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EXPERIMENTAL PROCEDURES |
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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 DH5Purification 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--D-galactopyranoside and
grown for an additional 2 h.
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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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 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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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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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.
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DISCUSSION |
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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 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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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