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Cloning and Characterization of the NADH Pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, Members of a Nudix Hydrolase Subfamily
Biochemical and Biophysical Research Communications 273, 753–758 (2000) doi:10.1006/bbrc.2000.2999, available online at http://www.idealibrary.com on
Cloning and Characterization of the NADH Pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, Members of a Nudix Hydrolase Subfamily WenLian Xu, Christopher A. Dunn, and Maurice J. Bessman 1 Department of Biology and the McCollum–Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218
Received May 31, 2000
Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli. The purified enzymes are homodimers of 39.1 and 43.5 kDa, respectively, are activated by Mg 2ⴙ and Mn 2ⴙ, and are 30 to 50 times more active on NADH than on NAD ⴙ. They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E. coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases. © 2000 Academic Press Key Words: NADH pyrophosphatase; Nudix hydrolase.
An important aspect of our systematic study of the Nudix hydrolase family of enzymes, is the identification of structural elements predictive of subfamilies whose members share the same or similar activities. The Nudix hydrolases are defined by the following array of amino acids: GX 5EX 7REUXEEXGU, where U is hydrophobic, usually Ile, Leu, or Val. At present, over 450 open reading frames containing this signature sequence, from over 85 species have been identified. The name, Nudix, is an acronym coined from the class of substrates hydrolyzed by these enzymes, predominantly nucleoside diphosphates linked to some other moiety, x. These compounds fall into several distinct classes including nucleotide sugars (1), diadenosine polyphosphates (2–9), (deoxy)nucleoside triphosphates (10 –16), and ADP-ribose (2, 17–19). Also, an enzyme with dual specificity active on the non-nucleotide substrate, diphosphoinositol polyphosphate, and in addition, diadenosine polyphosphates, has been described (20). Figure 1 is a partial list of open reading frames 1
To whom correspondence should be addressed. Fax: 410-5165213. E-mail: [email protected].
uncovered in a recent Blast (21) search for sequences homologous to the Nudix box. Since this same conserved amino acid array is present in all these putative enzymes acting on widely different nucleoside diphosphate derivatives, the individual specificity determinants must lie somewhere distal to the Nudix signature sequence. In a previous paper (19) we described two landmarks, a proline or tyrosine about 14 residues downstream of the Nudix box designating enzymes specific for ADP-ribose or dinucleoside polyphosphates respectively. We also hypothesized that an array of 8 conserved amino acids in this same region specify another subfamily of Nudix hydrolases active on NADH, of which the E. coli NADH pyrophosphatase is the prototype (22). In this paper we describe the cloning, expression, purification, and characterization of 2 Nudix hydrolases from Saccharomyces cerevisiae and Caenorhabditis elegans containing these 8 conserved amino acids, demonstrating that these proteins are, indeed, NADH pyrophosphatases, and that this unusual enzymatic activity is not confined to bacteria. MATERIALS AND METHODS Materials. Primers used were from Integrated DNA Technologies (Coralville, IA). Calf intestinal alkaline phosphatase was from Stratagene, and enzymes used in standard cloning procedures were from Life Technologies, Inc. and U.S. Biochemical Corp. Biochemicals were from Sigma. S. cerevisiae and common bacterial hosts were laboratory stocks, and C. elegans was supplied by Edward Hedgecock of this department. The plasmid, pGroESL, was a gift of George H. Lorimer, DuPont. Cloning. The gene corresponding to locus GLOGW (Accession No. z72589), coding for a 384-amino-acid open reading frame was amplified by PCR form S. cerevisiae genomic DNA prepared in this laboratory. The primers were designed to incorporate an NdeI restriction site at the start of the gene and a BamHI site at its termination, and the amplified gene product was ligated with predigested pET24a(⫹) containing a kanamycin resistance factor (Novagen). The resulting plasmid, pET24YstNADH was transformed into host BL21 (DE3) which had been transformed previously with pGroESL containing a chloramphenicol resistance marker. pGroESL expresses the chaper-
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FIG. 1. Representatives of the Nudix hydrolase family. A recent BLAST search (21) revealed more than 450 members of the Nudix hydrolase family from over 85 species. Shown here is a representative selection illustrating the highly conserved Nudix box in bold type.
onins GroES and GroEL helping to solubilize the pET 24YstNADH expressed protein. For preparation of the C. elegans homologue, RNA was prepared from worms according to Krause (23) and used to synthesize first strand cDNA with the Prostar, RT–PCR kit (Stratagene). This was used for standard PCR amplification of the target sequence for the putative NADH pyrophosphatase gene from C. elegans (Accession No. z68748). The amplified gene, engineered to contain NdeI and HindIII sites, was ligated into pET 24a(⫹) yielding pET 24CgsNADH which was transformed into BL21(DE3) containing pGroESL as above. Expression and purification of the enzyme. A single colony of either construct was inoculated into 10 ml of LB broth containing 30 g/ml each of Kanamycin and Chloramphenicol and grown at 37°C on a shaker overnight. The culture was diluted 50-fold in prewarmed, fresh medium and grown to an A 600 of 0.3 at which point it was transferred to a shaker at 22°C. When an A 600 of 0.9 was reached, the culture was induced in 0.5 mM isopropyl--Dthiogalactopyranoside and incubated overnight. Cells were har-
vested by centrifugation, washed in isotonic saline and either stored at ⫺80°C at this point, or suspended in 2.5 vol of 50 mM Tris–Cl, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol (TED buffer). Cells were ruptured in a French press, centrifuged to remove cellular debris, and the supernatant was adjusted to 10 mg/ml of protein with TED buffer. These crude extracts (Fraction I) for both enzymes were made 1.5% in streptomycin sulfate, the precipitates were removed by centrifugation and the supernatants represented Fraction II. For the S. cerevisiae orthologue, Fraction II was brought to 40% saturated ammonium sulfate and the precipitate was discarded. The ammonium sulfate concentration was increased to 50%, precipitating 95% of the enzyme, which was dissolved in a minimal volume of TED (Fraction III). This was loaded onto a gel filtration column (Superdex 75, 16/60, Pharmacia Biotech) previously equilibrated in TED plus 100 mM ammonium sulfate, and eluted with the same buffer. Active fractions were pooled, precipitated in 65% saturated ammonium sulfate, dissolved in TED buffer, dialyzed against TED plus 20% glycerol (Fraction IV), applied to a Mono Q anion exchange column (Pharmacia Biotech), and eluted with a 0.1–1 M gradient of
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FIG. 2. Alignment of the amino acid sequences from E. coli, S. cerevisiae, and C. elegans. The Nudix box is in bold type, and the downstream amino acids (highlighted in both bold type and dots) show the homology of the S. cerevisiae and C. elegans proteins, in this region, to the NADH pyrophosphatase of E. coli.
sodium chloride in TED plus 20% glycerol. Active fractions were pooled, concentrated with ammonium sulfate as above, dissolved in TED plus glycerol and stored at ⫺80°C (Fraction V). The C. elegans homologue followed a somewhat different purification scheme. Fraction II, the streptomycin supernatant, was treated with 40% saturated ammonium sulfate, precipitating most of the enzyme, which was dissolved in a minimal volume of TED (Fraction III) and chromatographed on a Sephadex G-100 gel-filtration column equilibrated and eluted with TED plus 200 mM sodium chloride. The active fractions were pooled (Fraction IV) applied to a hydroxyl apatite column and eluted with TED plus 0.1 M ammonium sulfate and 20% glycerol. Active fractions were pooled, concentrated, and stored as above (Fraction V). Enzyme assay. This assay measures the rate of conversion of an alkaline phosphatase resistant substrate, NADH, to phosphatase sensitive products, AMP and NMNH. The liberated inorganic orthophosphate was assayed colorimetrically. The standard reaction mixture contained in 50 l: 50 mM Tris–Cl, pH 7.5 for yeast or pH 8.5 for worm; 5 mM MgCl 2; 2.5 mM NADH; 4 units of alkaline intestinal phosphatase (EC 3.1.3.1) and 0.2–2 milliunits of enzyme. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 250 l of 4 mM EDTA, and P i was measured according to Ames and Dubin (24). A unit of enzyme hydrolyzes 1 mol of NADH per min under these conditions. Note that 2 mol of P i are formed for each mole of NADH hydrolyzed.
RESULTS AND DISCUSSION Gene cloning. The gene for yeast NADH pyrophosphatase was cloned directly from genomic DNA in
which no introns were present. Its sequence agreed exactly with that submitted to GenBank, Accession No. z72589. However, the orthologous gene from C. elegans was reported to have 5 introns. We therefore prepared cDNA and amplified the gene by PCR. Our nucleotide sequence was congruent with that reported for cosmid z68748, but our coding sequence differed in two respects. We found that nucleotides 19582–19599 represent an intron, not an exon as reported; likewise, nucleotides 20551–20659 represent an exon, not an intron. We attribute these differences to the fallibility of the Genefinder program. Protein expression and purification. Both the yeast and worm proteins expressed well from the plasmid constructs in the common host BL21 (DE3). However, almost all of the protein sedimented in a low speed centrifugation indicating that it was denatured. Accordingly, we modified our expression system by inducing the cells at a lower temperature, 22 instead of 37°C, and by including an expression plasmid for the GroESL chaperone. These modifications to the expression protocol had been successful in producing soluble protein in an otherwise refractory system (6) and they greatly improved the yield of soluble protein for both yeast and worm protein as well. The respective proteins were
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TABLE 3
Relative Specificities of the NADH Pyrophosphatases
Comparison of the NADH Pyrophosphatases
Enzyme
Substrate
C. elegans (%)
S. cerevisiae (%)
E. coli a (%)
NADH NAD ⫹ Ap 2A b Ap 4A b ADP-ribose GDP-mannose dATP dCTP dGTP dTTP
100 8 45 ⬍1 26 2 13 1 ⬍1 ⬍1
100 18 42 3 30 ⬍1 1 2 1 ⬍1
100 17 52 13 33 — 1 2 2 1
Introns Amino acids Molecular weight (kDa) Quaternary structure Metal requirement Substrate preference V max (units/mg)
Note. The activities were measured under standard assay conditions for each enzyme as described under Materials and Methods. When the nucleoside triphosphates were tested as substrates, the alkaline phosphatase was replaced by yeast inorganic pyrophosphatase (16). a These data for E. coli NADH pyrophosphatase are included for comparison and are taken from Frick et al. (22). b Ap 2A and Ap 4A are adenosine (5⬘) diphospho (5⬘) adenosine and adenosine (5⬘) tetraphospho (5⬘) adenosine, respectively.
purified in good yield to high purity (⬎90%) as assessed visually by gel electrophoresis (data not shown) following the procedures outlined in the methods section. Identification of enzymatic activity. Our usual approach to the identification of new members of the Nudix hydrolase family of enzymes is to test a variety of nucleoside diphosphate derivatives as potential substrates. However, in the present study, we hypothesized that the major substrate would be NADH, based on the conserved amino acids downstream of the Nudix box. An alignment of the yeast and the worm sequences along with the NADH pyrophosphatase of E. coli (22) is shown in Fig. 2. Note that only one small region outTABLE 2
Kinetic Analysis of the NADH Pyrophosphatases Enzyme S. cerevisiae
C. elegans
Parameters
NADH
NAD ⫹
NADH
NAD ⫹
V max (units a/mg) K cat (s ⫺1) K m (mM) K cat/K m (s ⫺1 M ⫺1)
12.3 8.9 1.6 5.8 ⫻ 10 3
0.95 0.69 6.6 0.1 ⫻ 10 3
6.6 4.3 1.4 3.1 ⫻ 10 3
0.97 0.63 6.6 0.1 ⫻ 10 3
Note. Rates were obtained under standard assay conditions by varying substrate concentrations. Data were analyzed with the software package “Enzyme Kinetics” from Trinity Software (Fort Pierce, FL). a A unit of activity is 1 mol of substrate hydrolyzed per minute.
S. cerevisiae
C. elegans
E. coli
0 385 43.5 Dimer Mg 2⫹, Mn 2⫹ NADH 12.3
5 345 39.1 Dimer Mg 2⫹, Mn 2⫹ NADH 6.6
0 257 29.7 Dimer Mg 2⫹, Mn 2⫹ NADH 7.6
side of the amino acids in the Nudix box is conserved in all three proteins. Accordingly, NADH was tested first, and it proved to be favored over all the nucleoside diphosphate derivatives known to be substrates for other members of the Nudix hydrolase family (17, 25). A representative list is shown in Table 1 along with the prototypical E. coli NADH pyrophosphatase for comparison. The striking aspect of the specificity is the marked preference for NADH over NAD ⫹ by both the worm and yeast enzymes, strongly reinforcing our earlier observation with the E. coli orthologue (22). Not only is there a 5- to 10 fold difference in rate of hydrolysis of NADH over NAD ⫹, but there is also a 5-fold lower K m for NADH as shown in Table 2. This factors into a 30- to 50-fold difference in catalytic efficiency (K cat/K m) for NADH over NAD ⫹. The reason for the preference for NADH over NAD ⫹ is not immediately obvious when activities toward other substrates are compared. For example, both Ap 2A and ADP-Ribose are better substrates than NAD ⫹ yet these analogues seem less structurally related to NADH than is NAD ⫹. In Ap 2A, nicotinamide is replaced by adenine, and in ADP-ribose, nicotinamide is missing altogether. This raises the possibility that the difference in reactivity of the two substrates is due to the positively charged pyridinium group in NAD ⫹ which may diminish its reactivity and/or binding at the active site of the enzyme. The credibility of this hypothesis will be tested when the three-dimensional structure of these enzymes is available. The crystal structure of the C. elegans enzyme is currently under investigation. 2 Other properties of the enzymes. The worm and yeast enzymes resemble the E. coli prototype in most respects. The products of the reaction are AMP and NMNH as determined by paper electrophoresis (26) and no inorganic orthophosphate is formed during the course of the reaction in the absence of alkaline phosphatase. A divalent cation is essential for activity. Mg 2⫹ at 5 mM is optimal and Mn 2⫹ at equal concentration is approximately 65% as effective. Zn 2⫹ and Ca 2⫹ 2
S. Gabelli, W-L. Xu, M. J. Bessman, and L. M. Amzel, unpublished results.
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FIG. 3. Predicted members of the NADH pyrophosphatase subfamily of the Nudix hydrolases. A recent BLAST search (21) identified 25 candidates for membership in the NADH pyrophosphatase subfamily of Nudix hydrolases. The first 3 (checked) have been positively identified previously (19, 22) or in this paper. The next 22 are predicted members on the basis of the homologous amino acids downstream of the Nudix box, set off by dots.
are not active and in the presence of Mg 2⫹ inhibit the enzyme completely. In keeping with most of the other Nudix hydrolases studied to date, the worm enzyme has a distinctly alkaline pH optimum of 8.5 whereas the yeast enzyme is uncharacteristically lower at 7.5. A comparison of the three enzymes is summarized in Table 3. It is evident that these 3 enzymes from widely divergent species are orthologues with similar structures and enzymatic activities. They were suspected of belonging to the same subfamily of Nudix hydrolases having NADH pyrophosphatase activity because of the conserved amino acids downstream of the Nudix box (19), and the experiments described in this paper establish them as such. A recent BLAST (21) search covering the region of the Nudix box and the downstream amino acids defining the NADH pyrophosphatases uncovered 22 more candidates for membership in this sub-family as shown in Fig. 3. The first three entries have been confirmed in this and the previous work (22), and the fourth entry almost certainly represents the human orthologue of NADH pyrophosphatase. Thus this highly unusual enzyme has been conserved from primitive to advanced species. We have deleted the gene from S. cerevisiae, but have seen no significant effects on growth under aerobic or anaerobic conditions or on survival of the mutant in stationary phase. The enzyme is the only known source of NMNH in the cell and as such may play some specialized
physiological role, or it may help maintain the NAD ⫹/ NADH ratio important in intermediary metabolism. The results reported in this paper establish the NADH pyrophosphatases as a bona fide subfamily of the Nudix hydrolases recognizable by a conserved array of amino acids downstream of the Nudix box. Two other subfamilies, the dinucleoside polyphosphatases and the ADP-ribose pyrophosphatases are distinguished by specific amino acids in this same region (19). Thus this broadly distributed family of Nudix hydrolases, defined by a specific amino acid signature sequence, and made up of different enzymes hydrolyzing different nucleoside diphosphate derivatives, may itself be subdivided into subfamilies of related enzymes whose activities are predictable from amino acid landmarks outside of the Nudix box. As we uncover more enzyme activities encoded by specific genes, we accumulate comparative data for predicting function from structure, which is the goal of functional genomics. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant GM 18649.
REFERENCES
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1. Frick, D. N., Townsend, B. D., and Bessman, M. J. (1995) J. Biol. Chem. 270, 24086 –24091.
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2. O’Handley, S. F., Frick, D. N., Dunn, C. A., and Bessman, M. J. (1998) J. Biol. Chem. 273, 3192–3197. 3. Thorne, N. M. H., Hankin, S., Wilkinson, M. C., Nunez, C., Barraclough, R., and McLennan, A. G. (1995) Biochem. J. 311, 717–721. 4. Hankin, S., Wintero, A. K., and McLennan, A. G. (1997) Int. J. Biochem. Cell Biol. 29, 317–323. 5. Maksel, D., Guranowski, A., Ilgoutz, S. C., Moir, A., Blackburn, M. G., and Gayler, K. R. (1998) Biochem. J. 329, 313–319. 6. Conyers, G. B., and Bessman, M. J. (1999) J. Biol. Chem. 274, 1203–1206. 7. Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604 – 8610. 8. Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649 –3655. 9. Cartwright, J. L., Britton, P., Minnick, M. F., and McLennan, A. G. (1999) Biochem. Biophys. Res. Commun. 256, 474 – 479. 10. Bhatnagar, S. K., Bullions, L. C., and Bessman, M. J. (1991) J. Biol. Chem. 266, 9050 –9054. 11. Mo, J.-Y., Maki, H., and Sekiguchi, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11021–11025. 12. Kakuma, T., Nishida, J.-I., Tsuzuki, T., and Sekiguchi, M. (1995) J. Biol. Chem. 270, 25942–25948. 13. Cai, J. P., Kakuma, T., Tsuzuki, T., and Sekiguchi, T. (1995) Carcinogenesis 16, 2343–2350.
14. Kamath, A. V., and Yanofsky, C. (1993) Gene 134, 99 –102. 15. Bullions, L. C., Me´jean, V., Claverys, J. P., and Bessman, M. J. (1994) J. Biol. Chem. 269, 12339 –12344. 16. O’Handley, S. F., Frick, D. N., Bullions, L. F., Mildvan, A. S., and Bessman, M. J. (1996) J. Biol. Chem. 271, 24649 –24654. 17. Sheikh, S., O’Handley, S. F., Dunn, C. A., and Bessman, M. J. (1998) J. Biol. Chem. 273, 20924 –20928. 18. Raffaelli, N., Lorenzi, T., Emanuelli, M., Ruggieri, S., and Magni, G. (1999) FEBS Lett. 444, 222–226. 19. Dunn, C. A., O’Handley, S. F., and Bessman, M. J. (1999) J. Biol. Chem. 274, 32318 –32324. 20. Yang, X., Safrany, S., and Shears, S. B. (1999) J. Biol. Chem. 274, 35434 –35440. 21. Altschul, S. F., Gish, W., Meyers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 203, 403– 410. 22. Frick, D. N., and Bessman, M. J. (1995) J. Biol. Chem. 270, 1529 –1534. 23. Krause, M. (1995) Methods Cell Biol. 48, 513–529. 24. Ames, B. N., and Dubin, D. T. (1960) J. Biol. Chem. 235, 769 – 775. 25. Bessman, M. J., Frick, D. N., and O’Handley, S. F. (1996) J. Biol. Chem. 271, 25059 –25062. 26. Markham, R., and Smith, J. D. (1952) Biochem. J. 52, 552– 557.
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Cloning and Characterization of the NADH Pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, Members of a Nudix Hydrolase Subfamily WenLian Xu, Christopher A. Dunn, and Maurice J. Bessman 1 Department of Biology and the McCollum–Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218
Received May 31, 2000
Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli. The purified enzymes are homodimers of 39.1 and 43.5 kDa, respectively, are activated by Mg 2ⴙ and Mn 2ⴙ, and are 30 to 50 times more active on NADH than on NAD ⴙ. They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E. coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases. © 2000 Academic Press Key Words: NADH pyrophosphatase; Nudix hydrolase.
An important aspect of our systematic study of the Nudix hydrolase family of enzymes, is the identification of structural elements predictive of subfamilies whose members share the same or similar activities. The Nudix hydrolases are defined by the following array of amino acids: GX 5EX 7REUXEEXGU, where U is hydrophobic, usually Ile, Leu, or Val. At present, over 450 open reading frames containing this signature sequence, from over 85 species have been identified. The name, Nudix, is an acronym coined from the class of substrates hydrolyzed by these enzymes, predominantly nucleoside diphosphates linked to some other moiety, x. These compounds fall into several distinct classes including nucleotide sugars (1), diadenosine polyphosphates (2–9), (deoxy)nucleoside triphosphates (10 –16), and ADP-ribose (2, 17–19). Also, an enzyme with dual specificity active on the non-nucleotide substrate, diphosphoinositol polyphosphate, and in addition, diadenosine polyphosphates, has been described (20). Figure 1 is a partial list of open reading frames 1
To whom correspondence should be addressed. Fax: 410-5165213. E-mail: [email protected].
uncovered in a recent Blast (21) search for sequences homologous to the Nudix box. Since this same conserved amino acid array is present in all these putative enzymes acting on widely different nucleoside diphosphate derivatives, the individual specificity determinants must lie somewhere distal to the Nudix signature sequence. In a previous paper (19) we described two landmarks, a proline or tyrosine about 14 residues downstream of the Nudix box designating enzymes specific for ADP-ribose or dinucleoside polyphosphates respectively. We also hypothesized that an array of 8 conserved amino acids in this same region specify another subfamily of Nudix hydrolases active on NADH, of which the E. coli NADH pyrophosphatase is the prototype (22). In this paper we describe the cloning, expression, purification, and characterization of 2 Nudix hydrolases from Saccharomyces cerevisiae and Caenorhabditis elegans containing these 8 conserved amino acids, demonstrating that these proteins are, indeed, NADH pyrophosphatases, and that this unusual enzymatic activity is not confined to bacteria. MATERIALS AND METHODS Materials. Primers used were from Integrated DNA Technologies (Coralville, IA). Calf intestinal alkaline phosphatase was from Stratagene, and enzymes used in standard cloning procedures were from Life Technologies, Inc. and U.S. Biochemical Corp. Biochemicals were from Sigma. S. cerevisiae and common bacterial hosts were laboratory stocks, and C. elegans was supplied by Edward Hedgecock of this department. The plasmid, pGroESL, was a gift of George H. Lorimer, DuPont. Cloning. The gene corresponding to locus GLOGW (Accession No. z72589), coding for a 384-amino-acid open reading frame was amplified by PCR form S. cerevisiae genomic DNA prepared in this laboratory. The primers were designed to incorporate an NdeI restriction site at the start of the gene and a BamHI site at its termination, and the amplified gene product was ligated with predigested pET24a(⫹) containing a kanamycin resistance factor (Novagen). The resulting plasmid, pET24YstNADH was transformed into host BL21 (DE3) which had been transformed previously with pGroESL containing a chloramphenicol resistance marker. pGroESL expresses the chaper-
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Representatives of the Nudix hydrolase family. A recent BLAST search (21) revealed more than 450 members of the Nudix hydrolase family from over 85 species. Shown here is a representative selection illustrating the highly conserved Nudix box in bold type.
onins GroES and GroEL helping to solubilize the pET 24YstNADH expressed protein. For preparation of the C. elegans homologue, RNA was prepared from worms according to Krause (23) and used to synthesize first strand cDNA with the Prostar, RT–PCR kit (Stratagene). This was used for standard PCR amplification of the target sequence for the putative NADH pyrophosphatase gene from C. elegans (Accession No. z68748). The amplified gene, engineered to contain NdeI and HindIII sites, was ligated into pET 24a(⫹) yielding pET 24CgsNADH which was transformed into BL21(DE3) containing pGroESL as above. Expression and purification of the enzyme. A single colony of either construct was inoculated into 10 ml of LB broth containing 30 g/ml each of Kanamycin and Chloramphenicol and grown at 37°C on a shaker overnight. The culture was diluted 50-fold in prewarmed, fresh medium and grown to an A 600 of 0.3 at which point it was transferred to a shaker at 22°C. When an A 600 of 0.9 was reached, the culture was induced in 0.5 mM isopropyl--Dthiogalactopyranoside and incubated overnight. Cells were har-
vested by centrifugation, washed in isotonic saline and either stored at ⫺80°C at this point, or suspended in 2.5 vol of 50 mM Tris–Cl, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol (TED buffer). Cells were ruptured in a French press, centrifuged to remove cellular debris, and the supernatant was adjusted to 10 mg/ml of protein with TED buffer. These crude extracts (Fraction I) for both enzymes were made 1.5% in streptomycin sulfate, the precipitates were removed by centrifugation and the supernatants represented Fraction II. For the S. cerevisiae orthologue, Fraction II was brought to 40% saturated ammonium sulfate and the precipitate was discarded. The ammonium sulfate concentration was increased to 50%, precipitating 95% of the enzyme, which was dissolved in a minimal volume of TED (Fraction III). This was loaded onto a gel filtration column (Superdex 75, 16/60, Pharmacia Biotech) previously equilibrated in TED plus 100 mM ammonium sulfate, and eluted with the same buffer. Active fractions were pooled, precipitated in 65% saturated ammonium sulfate, dissolved in TED buffer, dialyzed against TED plus 20% glycerol (Fraction IV), applied to a Mono Q anion exchange column (Pharmacia Biotech), and eluted with a 0.1–1 M gradient of
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FIG. 2. Alignment of the amino acid sequences from E. coli, S. cerevisiae, and C. elegans. The Nudix box is in bold type, and the downstream amino acids (highlighted in both bold type and dots) show the homology of the S. cerevisiae and C. elegans proteins, in this region, to the NADH pyrophosphatase of E. coli.
sodium chloride in TED plus 20% glycerol. Active fractions were pooled, concentrated with ammonium sulfate as above, dissolved in TED plus glycerol and stored at ⫺80°C (Fraction V). The C. elegans homologue followed a somewhat different purification scheme. Fraction II, the streptomycin supernatant, was treated with 40% saturated ammonium sulfate, precipitating most of the enzyme, which was dissolved in a minimal volume of TED (Fraction III) and chromatographed on a Sephadex G-100 gel-filtration column equilibrated and eluted with TED plus 200 mM sodium chloride. The active fractions were pooled (Fraction IV) applied to a hydroxyl apatite column and eluted with TED plus 0.1 M ammonium sulfate and 20% glycerol. Active fractions were pooled, concentrated, and stored as above (Fraction V). Enzyme assay. This assay measures the rate of conversion of an alkaline phosphatase resistant substrate, NADH, to phosphatase sensitive products, AMP and NMNH. The liberated inorganic orthophosphate was assayed colorimetrically. The standard reaction mixture contained in 50 l: 50 mM Tris–Cl, pH 7.5 for yeast or pH 8.5 for worm; 5 mM MgCl 2; 2.5 mM NADH; 4 units of alkaline intestinal phosphatase (EC 3.1.3.1) and 0.2–2 milliunits of enzyme. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 250 l of 4 mM EDTA, and P i was measured according to Ames and Dubin (24). A unit of enzyme hydrolyzes 1 mol of NADH per min under these conditions. Note that 2 mol of P i are formed for each mole of NADH hydrolyzed.
RESULTS AND DISCUSSION Gene cloning. The gene for yeast NADH pyrophosphatase was cloned directly from genomic DNA in
which no introns were present. Its sequence agreed exactly with that submitted to GenBank, Accession No. z72589. However, the orthologous gene from C. elegans was reported to have 5 introns. We therefore prepared cDNA and amplified the gene by PCR. Our nucleotide sequence was congruent with that reported for cosmid z68748, but our coding sequence differed in two respects. We found that nucleotides 19582–19599 represent an intron, not an exon as reported; likewise, nucleotides 20551–20659 represent an exon, not an intron. We attribute these differences to the fallibility of the Genefinder program. Protein expression and purification. Both the yeast and worm proteins expressed well from the plasmid constructs in the common host BL21 (DE3). However, almost all of the protein sedimented in a low speed centrifugation indicating that it was denatured. Accordingly, we modified our expression system by inducing the cells at a lower temperature, 22 instead of 37°C, and by including an expression plasmid for the GroESL chaperone. These modifications to the expression protocol had been successful in producing soluble protein in an otherwise refractory system (6) and they greatly improved the yield of soluble protein for both yeast and worm protein as well. The respective proteins were
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TABLE 3
Relative Specificities of the NADH Pyrophosphatases
Comparison of the NADH Pyrophosphatases
Enzyme
Substrate
C. elegans (%)
S. cerevisiae (%)
E. coli a (%)
NADH NAD ⫹ Ap 2A b Ap 4A b ADP-ribose GDP-mannose dATP dCTP dGTP dTTP
100 8 45 ⬍1 26 2 13 1 ⬍1 ⬍1
100 18 42 3 30 ⬍1 1 2 1 ⬍1
100 17 52 13 33 — 1 2 2 1
Introns Amino acids Molecular weight (kDa) Quaternary structure Metal requirement Substrate preference V max (units/mg)
Note. The activities were measured under standard assay conditions for each enzyme as described under Materials and Methods. When the nucleoside triphosphates were tested as substrates, the alkaline phosphatase was replaced by yeast inorganic pyrophosphatase (16). a These data for E. coli NADH pyrophosphatase are included for comparison and are taken from Frick et al. (22). b Ap 2A and Ap 4A are adenosine (5⬘) diphospho (5⬘) adenosine and adenosine (5⬘) tetraphospho (5⬘) adenosine, respectively.
purified in good yield to high purity (⬎90%) as assessed visually by gel electrophoresis (data not shown) following the procedures outlined in the methods section. Identification of enzymatic activity. Our usual approach to the identification of new members of the Nudix hydrolase family of enzymes is to test a variety of nucleoside diphosphate derivatives as potential substrates. However, in the present study, we hypothesized that the major substrate would be NADH, based on the conserved amino acids downstream of the Nudix box. An alignment of the yeast and the worm sequences along with the NADH pyrophosphatase of E. coli (22) is shown in Fig. 2. Note that only one small region outTABLE 2
Kinetic Analysis of the NADH Pyrophosphatases Enzyme S. cerevisiae
C. elegans
Parameters
NADH
NAD ⫹
NADH
NAD ⫹
V max (units a/mg) K cat (s ⫺1) K m (mM) K cat/K m (s ⫺1 M ⫺1)
12.3 8.9 1.6 5.8 ⫻ 10 3
0.95 0.69 6.6 0.1 ⫻ 10 3
6.6 4.3 1.4 3.1 ⫻ 10 3
0.97 0.63 6.6 0.1 ⫻ 10 3
Note. Rates were obtained under standard assay conditions by varying substrate concentrations. Data were analyzed with the software package “Enzyme Kinetics” from Trinity Software (Fort Pierce, FL). a A unit of activity is 1 mol of substrate hydrolyzed per minute.
S. cerevisiae
C. elegans
E. coli
0 385 43.5 Dimer Mg 2⫹, Mn 2⫹ NADH 12.3
5 345 39.1 Dimer Mg 2⫹, Mn 2⫹ NADH 6.6
0 257 29.7 Dimer Mg 2⫹, Mn 2⫹ NADH 7.6
side of the amino acids in the Nudix box is conserved in all three proteins. Accordingly, NADH was tested first, and it proved to be favored over all the nucleoside diphosphate derivatives known to be substrates for other members of the Nudix hydrolase family (17, 25). A representative list is shown in Table 1 along with the prototypical E. coli NADH pyrophosphatase for comparison. The striking aspect of the specificity is the marked preference for NADH over NAD ⫹ by both the worm and yeast enzymes, strongly reinforcing our earlier observation with the E. coli orthologue (22). Not only is there a 5- to 10 fold difference in rate of hydrolysis of NADH over NAD ⫹, but there is also a 5-fold lower K m for NADH as shown in Table 2. This factors into a 30- to 50-fold difference in catalytic efficiency (K cat/K m) for NADH over NAD ⫹. The reason for the preference for NADH over NAD ⫹ is not immediately obvious when activities toward other substrates are compared. For example, both Ap 2A and ADP-Ribose are better substrates than NAD ⫹ yet these analogues seem less structurally related to NADH than is NAD ⫹. In Ap 2A, nicotinamide is replaced by adenine, and in ADP-ribose, nicotinamide is missing altogether. This raises the possibility that the difference in reactivity of the two substrates is due to the positively charged pyridinium group in NAD ⫹ which may diminish its reactivity and/or binding at the active site of the enzyme. The credibility of this hypothesis will be tested when the three-dimensional structure of these enzymes is available. The crystal structure of the C. elegans enzyme is currently under investigation. 2 Other properties of the enzymes. The worm and yeast enzymes resemble the E. coli prototype in most respects. The products of the reaction are AMP and NMNH as determined by paper electrophoresis (26) and no inorganic orthophosphate is formed during the course of the reaction in the absence of alkaline phosphatase. A divalent cation is essential for activity. Mg 2⫹ at 5 mM is optimal and Mn 2⫹ at equal concentration is approximately 65% as effective. Zn 2⫹ and Ca 2⫹ 2
S. Gabelli, W-L. Xu, M. J. Bessman, and L. M. Amzel, unpublished results.
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FIG. 3. Predicted members of the NADH pyrophosphatase subfamily of the Nudix hydrolases. A recent BLAST search (21) identified 25 candidates for membership in the NADH pyrophosphatase subfamily of Nudix hydrolases. The first 3 (checked) have been positively identified previously (19, 22) or in this paper. The next 22 are predicted members on the basis of the homologous amino acids downstream of the Nudix box, set off by dots.
are not active and in the presence of Mg 2⫹ inhibit the enzyme completely. In keeping with most of the other Nudix hydrolases studied to date, the worm enzyme has a distinctly alkaline pH optimum of 8.5 whereas the yeast enzyme is uncharacteristically lower at 7.5. A comparison of the three enzymes is summarized in Table 3. It is evident that these 3 enzymes from widely divergent species are orthologues with similar structures and enzymatic activities. They were suspected of belonging to the same subfamily of Nudix hydrolases having NADH pyrophosphatase activity because of the conserved amino acids downstream of the Nudix box (19), and the experiments described in this paper establish them as such. A recent BLAST (21) search covering the region of the Nudix box and the downstream amino acids defining the NADH pyrophosphatases uncovered 22 more candidates for membership in this sub-family as shown in Fig. 3. The first three entries have been confirmed in this and the previous work (22), and the fourth entry almost certainly represents the human orthologue of NADH pyrophosphatase. Thus this highly unusual enzyme has been conserved from primitive to advanced species. We have deleted the gene from S. cerevisiae, but have seen no significant effects on growth under aerobic or anaerobic conditions or on survival of the mutant in stationary phase. The enzyme is the only known source of NMNH in the cell and as such may play some specialized
physiological role, or it may help maintain the NAD ⫹/ NADH ratio important in intermediary metabolism. The results reported in this paper establish the NADH pyrophosphatases as a bona fide subfamily of the Nudix hydrolases recognizable by a conserved array of amino acids downstream of the Nudix box. Two other subfamilies, the dinucleoside polyphosphatases and the ADP-ribose pyrophosphatases are distinguished by specific amino acids in this same region (19). Thus this broadly distributed family of Nudix hydrolases, defined by a specific amino acid signature sequence, and made up of different enzymes hydrolyzing different nucleoside diphosphate derivatives, may itself be subdivided into subfamilies of related enzymes whose activities are predictable from amino acid landmarks outside of the Nudix box. As we uncover more enzyme activities encoded by specific genes, we accumulate comparative data for predicting function from structure, which is the goal of functional genomics. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant GM 18649.
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