Analysis of the Catalytic and Binding Residues of the Diadenosine Tetraphosphate Pyrophosphohydrolase from Caenorhabditis elegans by Site-directed Mutagenesis*

Hend M. AbdelghanyDagger §, Scott Bailey||, G. Michael Blackburn**, John B. RaffertyDaggerDagger, and Alexander G. McLennanDagger §§

From the Dagger  School of Biological Sciences, Biosciences Building, University of Liverpool, P. O. Box 147, Liverpool L69 7ZB, United Kingdom, the  Department of Molecular Biology and Biotechnology, Firth Court, Sheffield S10 2TN, and the ** Department of Chemistry, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Sheffield S3 7HF, United Kingdom

Received for publication, November 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap4A hydrolase fusion proteins were expressed and their kcat and Km values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type kcat of 23 s-1 was reduced by 105-, 103-, and 30-fold, respectively, by replacement of the conserved P4-phosphate-binding catalytic residues Glu56, Glu52, and Glu103 by Gln. Km values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His31 to Val or Ala and Lys83 to Met produced 10- and 16-fold increases in Km compared with the wild type value of 8.8 µM. These residues stabilize the P1-phosphate. H31V and H31A had a normal kcat but K83M showed a 37-fold reduction in kcat. Lys36 also stabilizes the P1-phosphate and a K36M mutant had a 10-fold reduced kcat but a relatively normal Km. Thus both Lys36 and Lys83 may play a role in catalysis. The previously suggested roles of Tyr27, His38, Lys79, and Lys81 in stabilizing the P2 and P3-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr76 and Tyr121, which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased Km 4-fold. It is concluded that interactions with the P1- and P4-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that it may have a much wider substrate range then previously believed.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ap4A1 hydrolases are enzymes that hydrolyze dinucleoside polyphosphates. Structurally and mechanistically, they fall into two groups. The symmetrically cleaving enzymes (EC 3.6.1.41), such as Escherichia coli ApaH, generate 2-ADP from Ap4A, whereas the asymmetrically cleaving enzymes (EC 3.6.1.17) produce AMP and ATP (1, 2). The latter are members of the Nudix hydrolases, a family of structurally and catalytically similar enzymes that act upon a wide range of different nucleotide substrates. Some are highly specific whereas others appear to have a broad substrate range in vitro (3-5). The Nudix Ap4A hydrolases can be further subdivided into "plant" and "animal"-types, according to their primary structure (6). The plant-type includes enzymes from the Proteobacteria that have in some cases been shown to be associated with the invasion of mammalian cells, whereas the animal-type includes putative Ap4A hydrolases from Archaea (6-10). Early studies of both animal and plant Ap4A Nudix hydrolases employing a combination of substrate analogues and labeling with heavy isotopes of oxygen revealed the mechanism of hydrolysis to involve in-line nucleophilic attack of a water molecule at the P4 (Palpha ) phosphate with subsequent breakage of the P4-(O)P3 bond (8, 11-14). Recently, the catalytic residues of the lupin Ap4A hydrolase involved in this process were identified by a combination of structural analysis and site-directed mutagenesis (15-17). This study supported the catalytic mechanism previously described in detail for the prototypical Nudix hydrolase, the E. coli MutT 8-oxo-dGTPase.

Detailed structural studies of E. coli MutT first showed the importance of the highly conserved residues in the loop-helix-loop Nudix motif (Fig. 1). Glu53, Glu56, Glu57, Glu98 (outside the linear motif but structurally close), and the carbonyl of Gly38 coordinate an enzyme-bound Mg2+ ion. A water ligand of this ion is oriented or deprotonated for nucleophilic attack by Glu53, which is itself oriented by Arg52. A second metal ion is complexed to the substrate and neutralizes the charge on the attacked phosphate while Lys39 activates the NMP leaving group (18-21). The importance of Glu57 was indicated by a 105-fold reduction in kcat in a E57Q mutant (19). The contributions of the other residues to catalysis were also confirmed by site-directed mutagenesis: E53Q, E56Q, and E44Q led to 104.7-, 25-, and 14-fold decreases in kcat, respectively (20), whereas K39Q and R52Q produced 8-fold and >103-fold reductions, respectively (22). The principle of this catalytic mechanism appears to be well conserved among the Nudix hydrolases, including the lupin and Bartonella bacilliformis Ap4A hydrolases (15-17, 23), human MTH1 (24), yeast Dcp2p (25), and human NUDT3 (DIPP1) (26).


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Fig. 1.   The consensus Nudix motif and the actual motifs of E. coli MutT protein, C. elegans (C.e.) Ap4A hydrolase, and Lupinus angustifolius (L.a.) Ap4A hydrolase. The numbers indicate the positions in each primary structure.

Among the asymmetrically cleaving Ap4A hydrolases, identification of residues responsible for substrate binding as well as catalysis is of interest for two reasons. First, it will help our understanding of the evolution of substrate specificity among the Nudix hydrolases. Second, if the plant-type Ap4A hydrolase of invasive pathogenic bacteria is to be considered as a target for new antibacterial agents, the design of such agents will require knowledge of the subtle differences between the plant and animal types if selectivity is to be achieved. Recently we reported the crystal structure of an animal Ap4A hydrolase from the nematode Caenorhabditis elegans in both free form and after crystallization in the presence of the substrate analogue, AppCH2ppA (27, 28). The structure of the resulting binary complex allowed some predictions to be made about the importance of certain residues for substrate binding and catalysis and comparisons to be drawn with the lupin enzyme. Here, we extend these studies to include the effects of 19 site-specific mutations on Ap4A binding and hydrolysis by the C. elegans enzyme.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthesis of C. elegans First Strand cDNA Library-- Total RNA was isolated and purified from washed adult nematodes (C. elegans strain N2) using Trizol solution (Invitrogen) according to the manufacturer's instructions. Full-length C. elegans first strand cDNA was synthesized from this RNA using a first strand cDNA synthesis kit (MPI Fermentas). RNA (2 µl of 2.5 µg/µl) was added to 10 µl of RNase-free ddH2O. The solution was mixed gently, incubated at 70 °C for 5 min, and chilled on ice for 3 min before adding to a mixture containing 4 µl of 5× reaction buffer (250 mM Tris-HCl, pH 8.3, at 25 °C, 375 mM KCl, 15 mM MgCl2, 5 mM dithiothreitol), 1 µl of ribonuclease inhibitor (20 units/µl), 2 µl of oligo(dT)18 (0.5 µg/µl), 2 µl of dNTPs (10 mM each), and 2 µl of Moloney murine leukemia virus reverse transcriptase (20 units/µl, Promega). The reaction was incubated at 42 °C for 1 h, and then heated to 90 °C for 5 min. The library was stored at -20 °C.

Cloning of C. elegans Ap4A Hydrolase as a Glutathione S-Transferase (GST) Fusion Protein-- A cDNA corresponding to the C. elegans Y37H9A.6 Ap4A hydrolase gene (6) was amplified from the cDNA library by PCR using the forward and reverse primers d(CAGCGCCAGAATTCAATGGTCGTAAAAGCCGCGGG) and d(GAAATTACTCGAGAAAAATCGTTAAAATCCGGC), respectively. These primers provided an EcoRI restriction site at the start of amplified cDNA and a XhoI site at the end. After amplification with Taq DNA polymerase, the DNA was recovered, digested with EcoRI and XhoI, and the required restriction fragment ligated between the EcoRI and XhoI sites of the pGEX-6P-3 vector (Amersham Biosciences). The resulting construct, pGEX-Y37H9A, encoded the 137-amino acid Ap4A hydrolase fused to the C terminus of GST through a 6-amino acid linker.

Generation of Site-specific Mutants-- Site-directed mutagenesis was performed by PCR using the QuikChangeTM site-directed mutagenesis kit (Stratagene). PCR reactions contained pGEX-Y37H9A as template, Pfu Turbo DNA polymerase, and pairs of complementary oligonucleotide primers 37 to 43 nucleotides long containing the required mutations (Table I). Each reaction volume was 50 µl and contained the following: 50-100 ng of plasmid DNA, 125 ng of each mutagenic primer, 200 µM dNTPs, 10 mM KCl, 6 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 0.1% Triton X-100, 10 µg/ml bovine serum albumin, and 2.5 units of Pfu Turbo DNA polymerase. The PCR reaction protocol consisted of 2 min at 95 °C followed by 16 cycles of 95 °C for 1 min, 55 °C for 1 min, 68 °C for 14 min, followed by a final incubation at 72 °C for 15 min. Parental DNA was digested with 10 units of DpnI to degrade the methylated parental strands and the remaining plasmid DNA was used to transform E. coli XL1-Blue cells. For production of the Y76A/Y121A double mutant, the Y76A DNA construct was used as template in a PCR containing the Y121A mutagenic primers (Table I). The identities of all mutants were verified by complete sequencing of both DNA strands.

Expression and Purification of GST-Ap4A Hydrolase Fusion Proteins-- E. coli strain BL21(DE3) was transformed with pGEX-Y37H9A or its mutant derivatives. Cultures (250 ml) in LB medium containing 50 µg/ml ampicillin were grown to an A600 of 0.7 at 37 °C. Isopropyl-1-thio-beta -D-galactopyranoside was added to 1 mM and incubation continued for 2 h. Induced cells (approximately 1.6 g) were harvested by centrifugation at 10,000 × g, washed, and resuspended in 10 ml of ice-cold breakage buffer: 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane) (E-64, Sigma). Cell suspensions were sonicated and the resulting lysates cleared by centrifugation at 15,000 × g and 4 °C for 10 min. Supernatants were recovered and applied to columns containing 2.5 ml of glutathione-Sepharose 4B (Amersham Biosciences). Columns were washed with 25 ml of phosphate-buffered saline, followed by 25 ml of PreScission cleavage buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% (v/v) glycerol). Following complete elution of the buffer, the outlets were closed and 100 units of PreScission protease in 2.5 ml of cleavage buffer added to the resin and incubated for 18-20 h with gentle rocking at 4 °C. Cleavage of the GST domain from the Ap4A hydrolases was complete after 20 h. Columns were remounted, the resin left to settle, and the free Ap4A hydrolases containing the N-terminal extension GPLGSPNS eluted.

Ap4A Hydrolase Assay-- Ap4A hydrolase activity was measured using a luciferase-based bioluminescence assay as previously described (6). One ng enzyme protein was used in each case, except for K83M (10 ng), K79M (20 ng), E52Q and E103Q (60 ng), and E56Q (600 ng). This sensitive, continuous assay permits direct evaluation of initial rates. The increase in luminescence was linear for several minutes for each enzyme.

Other Methods-- Protein concentrations were estimated by the Coomassie Blue binding method (29) and protein molecular masses were determined by electrospray mass spectrometry as previously described (30).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of Wild Type and Mutant GST-Ap4A Hydrolase Fusion Proteins-- A cDNA corresponding to the C. elegans Y37H9A.6 Ap4A hydrolase gene was amplified from a cDNA library by PCR and inserted into the pGEX-6P-3 GST fusion vector to generate the recombinant plasmid pGEX-Y37H9A. When E. coli BL21(DE3) cells were transformed with this plasmid and induced with isopropyl-1-thio-beta -D-galactopyranoside, a major soluble 43-kDa band corresponding to the expected GST-Ap4A hydrolase fusion protein was detected (data not shown). The GST domain of this protein was readily removed by on-column cleavage with PreScission protease, resulting in the free Ap4A hydrolase with the N-terminal extension GPLGSPNS and mass 16.6 kDa.

Specific mutations were introduced into the Ap4A hydrolase coding region of pGEX-Y37H9A by PCR (Table I). The mutations were confirmed by DNA sequencing. A total of 16 single mutants and one double mutant involving 13 different residues was generated in this way. Their positions in the primary structure of the Ap4A hydrolase are shown in Fig. 2A and the locations of their alpha -carbon atoms in the three-dimensional structure of the binary complex are shown in Fig. 2B. Each was expressed as a GST fusion protein and purified after on-column cleavage and elution as described for the wild type. The predicted masses of the cleaved proteins were confirmed by mass spectrometry. With the exception of five (K36M, Y76A, Y76A/Y121A, K79M, and W32G) mutant proteins were substantially expressed in the soluble fraction (at least 40% of the total expressed recombinant protein) and the yields of purified proteins were nearly the same as for the wild type. The first four exceptions yielded about 5% of the recombinant protein in a soluble form, whereas W32G was completely insoluble when expressed. All kinetic data were determined using enzymes purified from the soluble fractions, which were all judged to be more than 95% pure by SDS-PAGE.

                              
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Table I
Oligonucleotide primers used for site-directed mutagensis of Ap4A hydrolase
Nucleotides that are noncomplementary to the template are underlined.


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Fig. 2.   Amino acid residues of the C. elegans Ap4A hydrolase changed by site-directed mutagenesis. A, the positions of residues in the amino acid sequence; conserved residues of the Nudix motif, residues which were mutated, and the resulting mutations are shown in bold. Numbers indicate the positions in the primary sequence. B, the locations of the alpha -carbon atoms of the mutated residues in the three-dimensional structure of the binary complex determined to a resolution of 1.8 Å (27) are indicated. The positions of the bound adenosine moiety (Ado), the P1- and P4-phosphates and the four bound Mg2+ ions are also shown.

Effects of Mutations on the Catalytic Properties of Ap4A Hydrolase-- The values of Km (8.8 µM) and kcat (23 s-1) estimated for the N-terminal extended wild type Ap4A hydrolase after cleavage from the GST domain were close to those previously reported for the native recombinant enzyme purified by conventional procedures (7.0 µM and 27 s-1, respectively) (6). From this we conclude that the 8-amino acid N-terminal extension does not interfere significantly with the binding of the substrate or with catalysis. The linearity of light output from the luminometric assay used was the same for all mutants as for the wild type, indicating the stability of the mutants under assay conditions. Km and kcat values were then determined for each mutant enzyme to determine the effects of each mutation on substrate binding and catalysis (Table II). For this enzyme, Km can be taken to approximate the dissociation constant of the ES complex(es), and hence as an inverse measure of affinity, based on the lack of effect of active site (kcat) mutants on the value of Km. Previous mutational studies with Nudix hydrolases have highlighted the importance of the Glu residues within the Nudix motif for catalysis (Fig. 1) (16, 20, 25, 26, 31, 32). Not surprisingly, therefore, the E56Q mutation was found to result in a 105-fold reduction in kcat and virtual abolition of detectable enzyme activity, exactly as was found for the equivalent residue (Glu59) in the lupin Ap4A hydrolase; in contrast, the Km was unaffected, indicating that the mutation has no effect on substrate binding. Similarly, neutralization of the charge on Glu52, the second of the three highly conserved Glu residues within the Nudix motif, by conversion to Gln (E52Q) reduced kcat by a factor of 103 but again had little effect on Km (Table II). On the basis of the 105-fold reduction in kcat, we previously proposed that Glu56 was most likely to be the catalytic base that deprotonates the attacking water molecule. However, the structural equivalents of Glu52 in the lupin Ap4A hydrolase (Glu55) and in the E. coli MutT protein (Glu53) have been proposed as the deprotonating base (11, 16, 20). Glu103, although not in the Nudix motif, is positioned close to it in the three-dimensional structure and coordinates two of the four Mg2+ ions located in the catalytic site (27). E103Q has a 30-fold lower kcat than the wild type and a similar Km. The equivalent mutations in E. coli MutT (E98Q) and the lupin Ap4A hydrolase (E125Q) produced 6.3- and 140-fold reductions in kcat, respectively (16, 20). Whereas these values suggest that Glu103 and its equivalents are unlikely to be the catalytic base in these enzymes, a detailed structural analysis of E. coli ADP-ribose pyrophosphatase has led to the conclusion that the equivalent Glu162 has this role in that enzyme (33, 34). Thus, although the architecture of the catalytic sites are broadly similar among the Nudix hydrolases, the mechanism of proton abstraction from the attacking water/hydroxyl appears to be subtly different in different family members.

                              
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Table II
Kinetic constants for Ap4A hydrolysis by the wild type and mutant Ap4A hydrolases
Values are the means of duplicate determination. In all cases, individual values differed from the mean by no more than 7.5%. Kinetic constants were determined directly by nonlinear regression analysis of the data.

Whereas several mutational studies of Nudix hydrolases have highlighted the importance to catalysis of particular residues in and adjacent to the catalytic motif, less attention has been focused on residues elsewhere that may be involved in substrate recognition and binding as well as catalysis. The crystal structure of the C. elegans Ap4A hydrolase binary complex showed that the adenine ring in the "AMP-binding pocket" distal to the P4-phosphate (the site of nucleophilic attack) was sandwiched between the phenolic rings of Tyr76 and Tyr121 and formed extensive pi -pi stacking interactions with these residues. To achieve this, a 90° rotation of the phenolic ring of Tyr121 about the chi 1 dihedral angle and an associated shift in Tyr76 occurred upon substrate binding (27). Both of these residues are highly conserved in structure-based sequence alignments of animal and plant Ap4A hydrolases. This, coupled with further interactions between the side chain hydroxyl group of Tyr121 and the 2'-OH of the attached ribose and between the ring of Tyr76 and the ribose O4 oxygen suggested that both Tyr residues should be important for substrate binding, therefore the effects of replacing each with Ala were investigated. As expected, both Y76A and Y121A showed an increased Km, but only by a factor of about 8 (Table II). Surprisingly, the combination of mutations in the double mutant Y76A/Y121A appeared to reduce the Km, again such that it was only 4-fold higher than the wild type. This suggests that the substrate may be able to bind effectively in a way that is independent of the Tyr residues (see "Discussion"). This alternative binding does lead to a lower catalytic rate, as evidenced by the reduced kcat values (20-fold less in the double mutant).

The crystal structure of the binary complex also showed that the P1-phosphate attached to the above adenosine moiety is stabilized on the enzyme via a series of hydrogen bonds/salt bridges between the phosphate oxygens and the side chain NZ nitrogens of Lys36 and Lys83, the side chain hydroxyl group of Tyr76, and the side chain imidazole ring Nepsilon 2 of His31 (27). Replacement of His31 with Ala (H31A) or Val (H31V) had a significant and specific impact on the binding of Ap4A, increasing the Km 8-12-fold while only marginally reducing kcat. Loss of the NZ nitrogen of Lys83 (K83M) led to an even greater increase in Km (16-fold) but in this case kcat was also substantially reduced (37-fold). The K36M mutant also had a reduced kcat (10-fold) but a relatively normal Km. These results confirm the predictions of the crystal structure and indicate the importance of Lys36 and Lys83, which is positioned such that it could also stabilize the P2-phosphate, to catalysis (27).

As there was no interpretable electron density for either the P2- or P3-phosphates in the binary complex, it was suggested that the side chains of His38 (within the Nudix motif) plus Lys79, Lys81, and Tyr27 (outside the Nudix motif) might be in appropriate positions to participate in P2- and P3-phosphate stabilization either by direct interaction or by metal coordination. The main chain amide of His38 is also the only direct protein contact with P4 via a hydrogen bond to one of the oxygen atoms (27). Potentially, His38 (structurally equivalent to Lys39 in the E. coli MutT protein) and/or Lys79 and/or Lys81 could neutralize the developing negative charge on the ATP leaving group, in much the same way as has been proposed for MutT Lys39 (18, 20). Therefore, appropriate mutants were generated to test these suggestions. Surprisingly, of the mutants analyzed (Y27A, Y27D, H38G, H38K, K79M, and K81M), only K79M showed a significant change in any kinetic constant, a substantial 140-fold reduction in kcat. However, Lys79, like Tyr27, is not well conserved among the Ap4A hydrolases, so this reduced activity may reflect a slight structural alteration in the protein rather than an important catalytic role. In contrast, Lys81 is well conserved as a basic residue in animal and plant Ap4A hydrolases. However, its mutation to Met resulted in a slight increase in kcat to 30 s-1, so it seems unlikely to be involved in stabilizing the leaving group. H38G and H38K also showed slight increases in kcat such that the kcat/Km ratio was 2.5-fold higher than the wild type. The equivalent residue in plant Ap4A hydrolases is a Gly, so, unlike Lys39 in MutT, His38 does not appear to be important for catalysis either. As all other residues in the region are too small to make contact, the conclusion is that there are few, if any, structurally or mechanistically important binding contacts for the P2- and P3-phosphates. This interesting point is discussed further below.

Finally, Trp32 is a completely conserved residue among Ap4A hydrolases of the Nudix family and is commonly found in other family members. This residue does not form interactions with the substrate but appears to stabilize the protein fold through interactions with Leu22 and Gln24 in the beta B strand and with Ile118 in the alpha II helix (27). Consistent with this essential structural role is the fact that the W32G mutant was completely insoluble and inactive when expressed.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The results of this analysis confirm the importance of residues previously implicated by structural analysis in binding and catalysis at the P4-phosphate and binding of the P1-phosphate of Ap4A by the C. elegans Ap4A hydrolase. It also provides important information on two further aspects of substrate binding by this enzyme that have implications for substrate recognition in the Nudix hydrolase family as a whole. First, there appear to be few, if any, important binding contacts for the P2- and P3-phosphates. Our previous structural analysis was unable to provide this information. It is possible that a nucleotide-bound metal ion is more important for stabilization of the negative charge during catalysis than any individual amino acid side chain. Such an ion appears to be required by the B. bacilliformis Ap4A hydrolase (23) and the E. coli ADP-ribose pyrophosphatase (33). However, the apparent absence of specific interactions in this region is entirely consistent with our recent discovery that this and related dinucleoside polyphosphate hydrolases can bind and hydrolyze 5-phosphoribosyl 1-pyrophosphate (35). In this case the ribose ring would occupy the P2,P3 site, with binding dependent solely on interactions in the P1 and P4 sites, in agreement with the data above.

Second, the mutational data relating to Tyr76 and Tyr121 were somewhat unexpected. The 90° rotation of the phenolic ring of Tyr121 in the binary complex compared with the apoenzyme and the pi -pi stacking interactions between both rings and the adenine ring of the adenosine moiety attached to the P1-phosphate originally suggested an essential role for these residues. However, substitution of both Tyr residues by Ala did not have the expected dramatic effect on substrate binding and yielded only a 4-fold increase in Km. Again, this is consistent with the finding that 5-phosphoribosyl 1-pyrophosphate, which lacks a base altogether, is a substrate. Thus, binding of one adenine ring between the Tyr residues is not an essential requirement for catalysis, although it undoubtedly contributes to the higher specificity constant for Ap4A compared with 5-phosphoribosyl 1-pyrophosphate (35). Interestingly, in the NMR structure of the lupin Ap4A hydrolase complexed with the substrate analogue ATP-MgFx, the adenine ring is not located between the structurally equivalent residues Tyr77 and Phe144, and instead Tyr77 is suggested to be important for the structural integrity of the enzyme-substrate complex rather than for direct substrate binding (17). Although structurally very similar, plant and animal Ap4A hydrolases typically show only 25-30% sequence similarity outside the Nudix motif and appear to form two distinct evolutionary groups within the family (6). Thus, either the animal and plant enzymes differ substantially in the way they bind substrate, or the possibility exists that Ap4A can bind to both Ap4A hydrolases in two different ways. Conceivably, one site represents the true substrate binding site before hydrolysis whereas the other is a transitional site for the ATP leaving group. Indeed, in view of the lack of electron density for the P2- and P3-phosphates and the second adenosine moiety in the crystal structure of the C. elegans binary complex, we have suggested that the analogue AppCH2ppA was probably hydrolyzed during crystallization (27). Thus, the visible AMP may be the AppCH2p product with its mobile P2- and P3-phosphates disordered in the crystal lattice and therefore invisible, or even the AMP product that has re-bound between the Tyr residues after departure of the ATP. If binding of the adenine ring between Tyr76 and Tyr121 is not essential, this could also explain our observations that phosphonate analogues with isopolar halomethylene groups bridging P1 and P2 and P3 and P4 such as ApCHFppCHFpA, and P1,P4-thiophosphates such as ApspppsA can be cleaved symmetrically by Nudix Ap4A hydrolases (14, 36). This requires attack at P2 or P3. Assuming that the loop-helix-loop structural motif containing the catalytic residues cannot move significantly, symmetrical hydrolysis implies that the substrate is bound in such a way as to present P3 rather than the usual P4 to the attacking nucleophile. Thus, substrates are able to bind in more than one location. Taken together, the ability of some Nudix hydrolases to use non-nucleotide substrates such as 5-phosphoribosyl 1-pyrophosphate and diphosphoinositol polyphosphates and the flexibility of substrate binding noted above suggest that the substrate range and function of Nudix hydrolases may be much wider than previously believed.

    FOOTNOTES

* This work was supported in part by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust (to J. B. R. and A. G. McL.).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.

§ Recipient of a postgraduate scholarship from the Egyptian government.

|| To whom correspondence should be addressed: School of Biological Sciences, Biosciences Building, University of Liverpool, P. O. Box 147, Liverpool L69 7ZB, United Kingdom. Tel.: 151-795-4426; Fax: 151-795-4404; E-mail: agmclen@liv.ac.uk.

Dagger Dagger Royal Society Olga Kennard Fellow.

§§ Present address: Dept. of Molecular Biophysics and Biochemistry, Yale University, Bass Center, 266 Whitney Ave., New Haven, CT 06520-8114.

Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M211983200

    ABBREVIATIONS

The abbreviations used are: Ap4A, diadenosine 5',5'''-P1,P4-tetraphosphate; AppCH2ppA, diadenosine 5',5'''-(P2,P3-methylene)-P1,P4-tetraphosphate; Nudix, nucleoside diphosphate linked to X; GST, glutathione S-transferase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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