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J. Biol. Chem., Vol. 277, Issue 18, 15317-15324, May 3, 2002

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Chlorella Virus RNA Triphosphatase

MUTATIONAL ANALYSIS AND MECHANISM OF INHIBITION BY TRIPOLYPHOSPHATE^*

Chunling Gong and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Received for publication, January 17, 2002, and in revised form, February 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu²⁴, Glu²⁶, Asp⁶⁴, Arg⁷⁶, Lys⁹⁰, Glu¹¹², Arg¹²⁷, Lys¹²⁹, Arg¹³¹, Asp¹⁴², Glu¹⁶³, and Glu¹⁶⁵) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K_i = 0.6 µM tripolyphosphate and 2.4 µM pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by three enzymatic reactions: (i) the 5' triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-7-)methyltransferase (1). The 5' ends of the mRNAs of DNA viruses such as papovaviruses, parvoviruses, adenoviruses, and herpesviruses are modified by the host cell's capping and methylating enzymes. However, other DNA viruses, including poxviruses, African swine fever virus (ASFV),¹ baculoviruses, and Chlorella virus PBCV-1, encode their own cap-forming enzymes.

Chlorella virus PBCV-1 is a large icosahedral DNA virus that replicates in unicellular Chlorella-like green algae (2). The 330-kb linear PBCV-1 genome encodes 375 polypeptides, which makes it one of the most complex viruses known. PBCV-1 encodes its own RNA triphosphatase and guanylyltransferase enzymes (3, 4). Chlorella virus guanylyltransferase is a 330-amino acid monomeric polypeptide that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. Its structure and mechanism have been elucidated by x-ray crystallography (5). Chlorella virus guanylyltransferase is monofunctional and has no intrinsic triphosphatase or methyltransferase activities. It is most closely related to the monofunctional yeast RNA guanylyltransferases, more so than to the multifunctional vaccinia virus and ASFV capping enzymes or the bifunctional triphosphatase-guanylyltransferases of baculoviruses.

Chlorella virus RNA triphosphatase (cvRtp1) is a 193-amino acid polypeptide that catalyzes metal-dependent hydrolysis of the -phosphate of triphosphate-terminated RNA (4). cvRtp1 is a member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatase components of the capping enzymes of fungi (Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe), protozoan and microsporidian parasites (Plasmodium falciparum, Trypanosoma brucei, and Encephalotozoon cuniculi), and metazoan DNA viruses (poxviruses, ASFV, and baculoviruses) (6-18) (Fig. 1). The signature biochemical property of this enzyme family is the ability to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of either manganese or cobalt. The defining structural features of the metal-dependent RNA triphosphatases are two glutamate-containing motifs (1 and 11 in Fig. 1) that are required for catalysis by the fungal, microsporidian, poxvirus, and baculovirus RNA triphosphatases. During our initial characterization of cvRtp1, we showed that alanine substitution of Glu²⁶ in the 1 motif (VELEFRLG) abrogated the phosphohydrolase activity (4).

The crystal structure of the S. cerevisiae RNA triphosphatase Cet1 revealed that the active site is located within a topologically closed hydrophilic tunnel composed of eight antiparallel  strands (Fig. 2) (19). The "triphosphate tunnel" has a distinctive architecture supported by an intricate network of hydrogen bonds and electrostatic interactions within the cavity, a high proportion of which are required for the triphosphatase activity of Cet1 (6, 11, 20). A single sulfate ion in the tunnel is coordinated by multiple basic side chains projecting into the cavity. It was proposed that the side chain interactions of the sulfate reflect contacts made by the enzyme with the -phosphate of the substrate (19). A manganese ion within the tunnel cavity is coordinated with octahedral geometry to a sulfate, to the side chain carboxylates of the two glutamates in 1, and to a glutamate in 11.

The sequence similarity between the Chlorella virus RNA triphosphatase and the catalytic domains of the S. cerevisiae, C. albicans, and S. pombe RNA triphosphatases, especially the  strands that comprise the triphosphate tunnel (Fig. 1), prompted a suggestion that the active site fold of the Chlorella virus and fungal RNA triphosphatases might be conserved as  barrels. In contrast, the RNA triphosphatases of poxviruses, ASFV, and baculoviruses have only limited primary structure similarity to the fungal and Chlorella virus enzymes. The poxvirus, ASFV, and baculovirus triphosphatases contain the diagnostic metal-binding motifs composed of alternating glutamate/hydrophobic side chains (the presumptive equivalents of strands 1 and 11 in the Cet1 structure), and the available mutational studies of the poxvirus and baculovirus proteins are consistent with an essential catalytic role for the conserved glutamates in metal binding (7, 8, 10). However, the poxvirus and baculovirus enzymes do not have obvious equivalents of the other six  strands that comprise the tunnel found in the fungal enzyme. The structural and mechanistic relatedness among the various DNA virus-encoded mRNA capping systems is of interest in light of the proposal that these diverse families of large DNA viruses (poxviruses, ASFV, and Chlorella virus) have a common evolutionary origin (24). Our hypothesis is that the Chlorella virus RNA triphosphatase is more closely related to the cellular triphosphatases of fungi than to the other DNA virus-encoded RNA triphosphatases.

To test the structural and mechanistic relatedness of the Chlorella virus and yeast RNA triphosphatases, we have conducted a mutational analysis of cvRtp1 using the tunnel of Cet1 as a guide. We used the alanine scanning approach to identify 12 new amino acids (in addition to Glu²⁶) that are essential or important for triphosphatase activity. The relevant structural features of these functional groups were then clarified by conservative amino acid substitutions. Our results underscore the fundamental similarities between the active sites of the fungal and Chlorella virus triphosphatases, but they also indicate that the functional contributions of some conserved residues are context-dependent. We show that tripolyphosphate is a potent competitive inhibitor of cvRtp1 that binds the enzyme with higher affinity than ATP. cvRtp1 has a weak tripolyphosphatase activity, indicating that a nucleoside is not strictly required for phosphohydrolase chemistry.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mutagenesis of cvRtp1-- Amino acid substitution mutations and diagnostic restriction sites were introduced into the cvRTP1 gene (viral open reading frame A449R) by the two-stage overlap extension method (21). pET-A449R (5) was used as the template for the first-stage amplification. The mutated full-length cvRTP1 genes generated in the second-stage amplification were digested with NdeI and BamHI and then inserted into pET16b. The insert of the resulting pET-cvRTP plasmids was sequenced completely to confirm the desired mutations and exclude the acquisition of unwanted changes during amplification or cloning. The pET-cvRTP plasmids were introduced into Escherichia coli BL21(DE3).

Expression and Purification of Recombinant cvRtp1-- Cultures (250 ml) of E. coli BL21(DE3)/pET-cvRTP were grown at 37 °C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A₆₀₀ reached ~0.5. The cultures were placed on ice for 10 min and then adjusted to 0.4 mM isopropyl-1-thio--D-galactopyranoside and 2% (v/v) ethanol. After further incubation for 17 h at 18 °C with constant shaking, the cells were harvested by centrifugation, and the pellets were stored at 80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 20 ml of buffer A (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 10% sucrose). Lysozyme was added to a final concentration of 50 µg/ml, and the suspension was incubated on ice for 15 min and then adjusted to 0.1% Triton X-100 and sonicated to reduce the viscosity of the lysate. Insoluble material was removed by centrifugation. The soluble extracts were applied to 0.75-ml columns of Ni²⁺-NTA-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100. The columns were washed with the same buffer and then eluted stepwise with buffer B (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10% glycerol, and 0.1% Triton X-100) containing 0, 50, 100, 200, 500, and 1000 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The wild-type and mutant cvRtp1 proteins were retained on the column and recovered predominantly in the 200 mM imidazole fractions (0.5-1 mg of protein). The Ni-agarose enzyme preparations were stored at 80 °C. Protein concentrations were determined using the Bio-Rad dye reagent with bovine serum albumin as a standard.

Nucleoside Triphosphate Phosphohydrolase Assay-- Reaction mixtures (10 µl) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1 mM MnCl₂, 0.2 mM [-³²P]ATP, and serial 2-fold dilutions of either wild-type or mutant cvRtp1 proteins (6.25, 12.5, 25, 50, 100, and 200 ng) were incubated for 15 min at 37 °C. The reactions were quenched by adding 2.5 µl of 5 M formic acid. Aliquots of the mixtures were applied to polyethyleneimine-cellulose TLC plates, which were developed with 1 M formic acid and 0.5 M LiCl. ³²P_i release was quantitated by scanning the chromatogram with a Fujix phosphorimager. Two separate titration experiments were performed for each mutant protein, and the average values of ³²P_i release were plotted as a function of input cvRtp1. The specific activities were calculated from the slopes of the titration curves. The activity values for the mutant enzymes were then normalized to the specific activity of wild-type cvRtp1.

Tripolyphosphatase Assay-- Reaction mixtures (50 µl) containing 50 mM Tris-HCl, pH 8.0, 5 mM DTT, 1 mM MnCl₂, 0.2 mM tripolyphosphate (or pyrophosphate or ATP), and cvRtp1 as specified were incubated for 15 min at 37 °C. The reactions were quenched by adding 1 ml of malachite green reagent (BIOMOL GREEN reagent, purchased from BIOMOL Research Laboratories, Plymouth Meeting, PA). Phosphate release was determined by measuring A₆₂₀ and extrapolating the value to a phosphate standard curve. Background levels of inorganic phosphate (typically 2-4% of the input levels of PPP_i, PP_i, or ATP substrate) determined from the A₆₂₀ of a control reaction mixture lacking cvRtp1 were subtracted from the values measured for the reactions containing cvRtp1.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Structure-based Alanine Scan of cvRtp1-- The crystal structure of S. cerevisiae Cet1 reveals that the active site is located within a topologically closed eight-stranded  barrel referred to as the "triphosphate tunnel" (19). The  strands of the tunnel (1, 5, 6, 7, 8, 9, 10, and 11) are displayed over the Cet1 amino acid sequence shown in Fig. 1. Most of the hydrophilic amino acids that comprise the active site of yeast RNA triphosphatase (Fig. 2) and are important for its catalytic activity (6, 11, 20, 22) are also present in cvRtp1. To elucidate structure-activity relationships for cvRt1p, we tested the effects of single alanine mutations at the 16 conserved side chains indicated by dots in Fig. 1. These were: Glu²⁴, Arg²⁸, Asp⁶⁴, Arg⁷⁶, Lys⁹⁰, Glu¹¹², Arg¹²⁷, Lys¹²⁹, Arg¹³¹, Thr¹³³, Lys¹⁴⁰, Asp¹⁴², Thr¹⁵⁹, Glu¹⁶¹, Glu¹⁶³, and Glu¹⁶⁵. At least one amino acid was mutated in each of the putative homologs of the  strands that comprise the Cet1 triphosphate tunnel. The Ala mutations were introduced into the cvRTP1 gene, and the cvRtp1-Ala polypeptides were expressed as His₁₀-tagged derivatives in E. coli in parallel with wild-type cvRtp1. The recombinant proteins were purified from soluble bacterial extracts by nickel-agarose chromatography. SDS-PAGE analysis of the polypeptide compositions of the nickel-agarose protein preparations revealed similar extents of purification (Fig. 3). cvRtp1 was the predominant polypeptide in every case.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Structural similarity between fungal, Plasmodium, and Chlorella virus RNA triphosphatases. The amino acid sequence of the catalytic domain of S. cerevisiae RNA triphosphatase Cet1 is aligned to the sequences of C. albicans CaCet1, S. cerevisiae Cth1, S. pombe Pct1, P. falciparum Prt1, and Chlorella virus cvRtp1. Gaps in the alignment are indicated by dashes. Poly-asparagine inserts in P. falciparum Prt1 are omitted from the alignment and are denoted by . The  strands that form the triphosphate tunnel of S. cerevisiae Cet1 are denoted above the sequence. Peptide segments with the highest degree of conservation in all six proteins are highlighted by the shaded boxes. Hydrophilic amino acids of cvRtp1 that were targeted for mutational analysis are denoted by dots below the sequence.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Structure of the active site of S. cerevisiae RNA triphosphatase. Stereo view of a cross section of the triphosphate tunnel of Cet1 (Protein Data Bank ID code 1D8I). The figure highlights the network of bonding interactions, especially those that coordinate sulfate (-phosphate) and manganese. The manganese interacts with octahedral geometry with the sulfate, three glutamates, and two waters. A putative nucleophilic water is coordinated by Glu433.

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Alanine mutants of cvRtp1. Aliquots (4 µg) of the Ni2+-agarose preparations of wild-type (WT) cvRtp1 and the indicated alanine mutants were analyzed by SDS-PAGE. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kDa) of marker proteins are indicated on the left.

Effects of Alanine Mutations on cvRtp1 Triphosphatase Activity-- The triphosphatase activities of the wild-type and mutant cvRtp1 proteins were assayed by the release of ³²P_i from 0.2 mM [-³²P]ATP in the presence of 1 mM manganese. The specific activities of the 16 Ala mutants, normalized to the wild-type specific activity, are shown in Fig. 4. Alanine substitutions at 9 of the 16 positions examined elicited at least a 100-fold decrement in catalytic activity. These residues were: Glu²⁴, Asp⁶⁴, Arg⁷⁶, Lys⁹⁰, Glu¹¹², Arg¹²⁷, Lys¹²⁹, Arg¹³¹, and Glu¹⁶³. Severe mutational effects were also observed for mutants D142A (4% of wild-type activity) and E165A (1.5% of wild type activity). We designated these 11 residues as essential for the phosphohydrolase activity of cvRtp1 by applying a criterion for essentiality of a 20-fold activity decrement incurred by side chain removal. Residues at which alanine substitution reduced activity to 6-20% of wild-type (e.g. Glu¹⁶¹ in 11) were deemed to be important but not essential, whereas residues at which alanine substitutions elicited less than a 5-fold effect (Arg²⁸, Thr¹³³, Lys¹⁴⁰, and Thr¹⁵⁹) were judged not to be important for triphosphatase activity.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of alanine substitutions on cvRtp1 activity. The ATPase specific activities of 16 new cvRtp1 alanine mutants were calculated from the slopes of the titration curves and then normalized to the specific activity of wild-type cvRtp1 (defined as 100%). The activity of mutant E26A (denoted by an asterisk) was reported previously (4). The amino acid positions of S. cerevisiaie Cet1 and C. albicans CaCet1 that correspond to the 17 mutated residues of cvRtp1 (as judged from the alignment in Fig. 1) are indicated to the right, along with the previously reported ATPase specific activities of the indicated alanine mutants of Cet1 and CaCet1, which are normalized to the respective wild-type ATPase activities (6, 11, 13, 20).

Comparison of Alanine Mutation Effects on Chlorella virus, S. cerevisiae, and C. albicans Triphosphatases-- Fig. 4 shows a comparison of the effects of alanine mutations at the "equivalent" amino acids of cvRtp1, S. cerevisiae Cet1, and C. albicans CaCet1. The figure lists the specific activities of the Cet1-Ala and CaCet1-Ala proteins in manganese-dependent hydrolysis of ATP (6, 11, 13, 20). All of the 16 mutants generated in cvRtp1 have counterparts in Cet1, and 14 of the 16 corresponding mutants have been analyzed in CaCet1. The salient point of the comparison is that most of the mutational effects are concordant in all three metal-dependent phosphohydrolases. Nonetheless, disparities are noted at four positions of cvRtp1 versus Cet1 (these are Asp⁶⁴, Lys⁹⁰, Arg¹²⁷, and Lys¹⁴⁰ in cvRtp1). The mutational data can be interpreted in light of the crystal structure of Cet1.

We have grouped the essential and important active site residues of Cet1 into three functional classes (20). Class I residues of Cet1 participate directly in catalysis via coordination of the -phosphate (Arg³⁹³ in 6 and Lys⁴⁵⁶ and Arg⁴⁵⁸ in 9) or the essential metal (Glu³⁰⁵ and Glu³⁰⁷ in 1 and Glu⁴⁹⁶ in 11) (Fig. 2). All six amino acids in this category are essential for the triphosphatase activities of both cvRtp1 and CaCet1. We surmise, therefore, that the equivalents of the class I residues in cvRtp1 (Arg⁷⁶, Lys¹²⁹, Arg¹³¹, Glu²⁴, Glu²⁶, and Glu¹⁶⁵) are probably involved in coordination of the -phosphate and the metal.

Class II residues of Cet1 make water-mediated contacts with the -phosphate (Asp³⁷⁷ in 5 and Glu⁴³³ in 8) or the metal (Asp⁴⁷¹ in 10 and Glu⁴⁹⁴ in 11). The two residues that indirectly coordinate the metal are essential for cvRtp1 and CaCet1. The conserved glutamate in 8 that coordinates via water to the phosphate is also essential in cvRtp1 and CaCet1. However, the conserved aspartate in 5 is evidently more critical for the function of cvRtp1 (D64A has 0.9% of wild-type triphosphatase activity) than it is for either Cet1 (D377A has 8% activity) or CaCet1 (D363A has 12% activity).

Class III residues of Cet1 function indirectly in catalysis via their interactions with other essential side chains and/or their stabilization of the tunnel architecture. These residues include Lys⁴⁰⁹ in 7, Arg⁴⁵⁴ in 9, Arg⁴⁶⁹ in 10, and Glu⁴⁹² in 11. It is in this functional category that the Chlorella virus and fungal enzymes displayed the greatest differences in alanine substitution effects. For example, the K409A mutation reduced the activity of Cet1 to 11%, whereas the equivalent mutations in cvRtp1 and CaCet1 abolished their triphosphatase activity (Fig. 4). The R454A mutation of Cet1 (15% activity) was also less deleterious than the equivalent R127A mutation in cvRtp1 (0.7% activity) and the R441A mutation in CaCet1 (3% activity). On the other hand, whereas Arg⁴⁶⁹ was essential for Cet1 (R469A has 3% of wild-type activity), the loss of the basic Lys¹⁴⁰ side chain at the equivalent position of cvRtp1 had only a modest effect on activity (37% of wild-type) that was judged not to be catalytically significant. In the crystal structure of Cet1, Arg⁴⁶⁹ is located on the exit side of the tunnel and is not in proximity to the -phosphate. Thus, it was proposed that Arg⁴⁶⁹ is not a direct catalyst and that its essentiality for Cet1 reflects its role in positioning Asp⁴⁷¹, which is essential. Based on the present findings for cvRtp1, we conclude that a basic residue in 10 is not inevitably required to position the neighboring Asp in 10 for its role in water-mediated metal binding. Thus, the role for the basic residue is context-dependent. The equivalent lysine residue in CaCet1 has not been subjected to mutational analysis. This position is occupied by histidine or glutamine in other members of the metal-dependent phosphohydrolase family (Fig. 1). It is therefore noteworthy that introducing a lysine or glutamine in place of Arg⁴⁶⁹ in S. cerevisiae Cet1 reduced triphosphatase activity to 1% of the wild-type value (20). The essential role of arginine at this position appears to be unique to the S. cerevisiae enzyme.

Arg²⁸ in 1 of cvRtp1 is conserved as a lysine or arginine in all of the triphosphatases shown in Fig. 1. In the Cet1 crystal structure, the equivalent residue Lys³⁰⁹ coordinates the essential Glu³⁰⁷ in 1. Here we found that Arg²⁸ plays no apparent role in catalysis by cvRtp1, which agrees with previous findings for Lys³⁰⁹ in Cet1 and Lys²⁹¹ in CaCet1 (Fig. 4). Thr¹³³ in 9 of cvRtp1 is conserved as a serine or threonine in five of the six triphosphatases shown in Fig. 1, yet alanine mutations at this position had no significant effect on cvRtp1 or Cet1 triphosphatase activity (Fig. 4). Thr¹⁵⁹ in 11 of cvRtp1 corresponds to Thr⁴⁹⁰ of Cet1, and neither side chain is functionally significant; this is in keeping with the lack of conservation at this position in other members of the triphosphatase family.

Structure-Activity Relationships at Essential and Important Amino Acids of cvRtp1-- Conservative substitutions were introduced at each of the 12 residues defined by the present alanine scan as essential or important for cvRtp1 function. We also introduced conservative substitutions for Glu²⁶, which had been previously shown to be essential for activity (4). A total of 24 recombinant proteins with conservative changes were produced in E. coli and purified from soluble bacterial extracts by nickel-agarose chromatography (Fig. 5). The manganese-dependent ATPase activities of the conservative mutants were determined by protein titration, and the specific activity values, normalized to that of wild-type cvRtp1, are shown in Fig. 6.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Conservative mutants of cvRtp1. Aliquots (4 µg) of the Ni2+-agarose preparations of wild-type (WT) cvRtp1 and the indicated mutants were analyzed by SDS-PAGE. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kDa) of marker proteins are indicated on the left and right.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effects of conservative substitutions on cvRtp1 activity. The ATPase specific activities of 24 conservative cvRtp1 mutants were calculated from the slopes of the titration curves and then normalized to the specific activity of wild-type cvRtp1 (defined as 100%). The effects of conservative substitutions at the equivalent positions of Cet1 on ATPase specific activity (expressed as the percentage of the wild-type ATPase activity) are indicated to the right.

Substitution of any of the three essential arginines of cvRtp1 (Arg⁷⁶, Arg¹²⁷, and Arg¹³¹) by lysine failed to restore the triphosphatase activities to >3% of the wild-type level and resulted in only marginal recovery above the activity of the respective arginine-to-alanine mutants. We surmise that cvRtp1 function requires a bidentate arginine side chain at each position and not merely positive charge. The essentiality of the bidentate arginines Arg⁷⁶ and Arg¹³¹ in cvRtp1 is in accord with the inability of lysine to substitute for the corresponding 6 and 9 arginines in Cet1 (Arg³⁹³ and Arg⁴⁵⁸), whereas the effects of conservative mutations in cvRtp1 Arg¹²⁷ versus Cet1 Arg⁴⁵⁴ are evidently discordant (Fig. 6).

The two essential lysines (Lys⁹⁰ and Lys¹²⁹) were replaced by arginine and glutamine. The K90R and K90Q proteins were severely defective, with 0.2% and 0.1% of wild-type activity, respectively. The R129K change restored a low level of activity over that of the alanine mutant (3.9% versus 0.2%), but the R129Q mutation had no salutary effect. Thus, cvRtp1 triphosphatase activity specifically requires a lysine at positions 90 and 129. Similar strict requirements for lysine pertain at the corresponding Cet1 residues Lys⁴⁰⁹ (7) and Lys⁴⁵⁶ (9) (Fig. 6).

The two essential aspartic acids of cvRtp1 (Asp⁶⁴ and Asp¹⁴²) were replaced by glutamate and asparagine. The D142E and D142N proteins had 3.5% and 1% of wild-type activity, respectively, similar to the 4% activity of the D142A mutant. We surmise that ATPase activity is strictly dependent on a carboxylate functional group and that steric constraints preclude restoration of function by the longer glutamate side chain. Similar structure-activity relationships were observed for the corresponding Asp⁴⁷¹ position of Cet1 (Fig. 6). Introduction of a glutamate in lieu of Asp⁶⁴ restored function to 50% of wild-type specific activity, compared with 0.9% activity for the D64A mutant. The D64N change also restored activity, albeit only to ~9% of the wild-type level. We surmise that optimal activity depends on a carboxylate functional group at position 64 of cvRtp1 and that the active site can accommodate the longer glutamate side chain. The partial activity conferred by the amide group of asparagine suggests that this residue makes critical hydrogen bonding interactions, conceivably with a water coordinated to the -phosphate, as in the structure of Cet1 (Fig. 2). Note that the structure-activity relationships at this position differ for cvRtp1 and Cet1. The aspartic acid is important for Cet1 activity (but not essential as in cvRtp1), and the Cet1 ATPase was restored completely when Asp³⁷⁷ was replaced with either glutamate or asparagine (Fig. 6).

The three essential glutamates of cvRtp1 (Glu²⁴, Glu²⁶, and Glu¹⁶⁵) corresponding to the three metal-binding side chains of Cet1 (Glu³⁰⁵, Glu³⁰⁷, and Glu⁴⁹⁶) were substituted conservatively by glutamine and aspartate. The E24D, E24Q, E26D, and E26Q changes were just as deleterious as the respective alanine mutations (Fig. 6). These findings are concordant with the conservative mutational effects for Cet1, and they suggest that Glu²⁴ and Glu²⁶ of cvRtp1 are likely to coordinate the divalent metal directly and that cvRtp1 cannot flex its structure to bring an aspartate (with its shorter main chain to carboxylate linker) into the metal coordination sphere. Replacing Glu¹⁶⁵ of cvRtp1 with aspartate had no salutary effect compared with the alanine mutant, whereas introducing glutamine elicited a partial restoration of function to 10% of wild-type activity (Fig. 6). The partial activity of the E165Q mutant presumably reflects the importance of hydrogen-bonding interactions of this side chain. Different structure-activity relationships were obtained for the corresponding Glu⁴⁹⁶ of Cet1, where neither aspartate nor glutamine revived the ATPase activity.

The essential function of Glu¹⁶³ of cvRtp1 could not be satisfied by either glutamine (<0.1% of wild-type activity) or aspartic acid (3% activity) (Fig. 6). In contrast, the function of the corresponding Glu⁴⁹⁴ side chain of Cet1, which interacts with a water molecule coordinated by the enzyme-bound metal (Fig. 2), was restored by aspartic acid (to 24% of wild-type activity), albeit not by glutamine. Thus, Cet1 is more tolerant than cvRtp1 of a retraction of the main chain to carboxylate distance at this position. The Glu¹⁶¹ side chain of cvRtp1 was classified as important based on the 13% residual activity of the E161A mutant. The corresponding Glu⁴⁹² residue in Cet1 forms a salt bridge with Arg⁴⁵⁴ (equivalent to essential residue Arg¹²⁷ in cvRtp1), which is postulated to stabilize the tunnel architecture (Fig. 2). The conservative E161D mutation of cvRtp1 (0.7% activity) was more deleterious that the E161A change, whereas the E161Q change resulted in a substantial gain of function to 63% of the wild-type level (Fig. 6). The high activity afforded by the amide group indicates that a putative salt bridge to Arg¹²⁷ is not essential and implies that hydrogen-bonding interactions may suffice to properly stabilize the fold of the active site and orient the Arg¹²⁷ side chain for interaction with the substrate. Retraction of the carboxylate closer to the main chain (in E161D) evidently imposed harmful steric or electrostatic clashes on cvRtp1 that did not arise when the equivalent E492D change was introduced into Cet1.

Finally, it was instructive that the essential function of Glu¹¹² in cvRtp1 could not be fulfilled by aspartate (0.7% activity), whereas substitution with glutamine restored phosphohydrolase activity to ~10% of the wild-type level (Fig. 6). The corresponding Glu⁴³³ side chain of Cet1 coordinates a water molecule that is in turn coordinated by the sulfate (-phosphate) at the active site. It was proposed previously that this water is the nucleophile that attacks the  phosphorus and that Glu⁴³³ acts as a general base catalyst to activate the nucleophilic water (20). This model is consistent with the finding that replacement of Glu⁴³³ with glutamine, which cannot abstract a proton from water, resulted in a 100-fold decrement in Cet1 phosphohydrolase activity. The partial activity of the E112Q mutant of cvRtp1 suggests subtle differences in the catalytic mechanism of cvRtp1 versus Cet1, i.e. that orientation of the putative nucleophilic water via hydrogen-bonding interactions (e.g. to an amide) enhances cvRtp1 activity 10-fold, whereas proton transfer to the glutamate, if applicable, contributes only a 10-fold stimulation of activity.

Inhibition of cvRtp1 by Tripolyphosphate and Pyrophosphate-- We tested the effects of various phosphate derivatives on the ability of cvRtp1 to hydrolyze 0.2 mM [-³²P]ATP in the presence of 1 mM manganese. Inorganic phosphate, which is a product of the phosphohydrolase reaction, inhibited activity by 30% at 0.5 mM P_i and by 50% at 0.7 mM P_i (Fig. 7; data not shown). Inorganic pyrophosphate was a much more potent inhibitor than phosphate (50% inhibition at 30 µM PP_i), whereas tripolyphosphate was even more potent than pyrophosphate (50% inhibition at 4 µM PPP_i) (Fig. 7). The findings that tripolyphosphate and pyrophosphate elicited 50% inhibition when present at concentrations 25-fold and 7-fold less than input ATP and 250-fold and 33-fold less than input manganese argue against the possibility that these agents inhibit by acting as chelators to compete with ATP (or enzyme) for the metal cofactor. A simple explanation for the potent inhibition is that PPP_i and PP_i bind more avidly than ATP to the triphosphate-binding pocket within the active site tunnel.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Inhibition of cvRtp1 ATPase activity by inorganic phosphate, pyrophosphate, and tripolyphosphate. Reaction mixtures (10 µl) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1 mM MnCl2, 0.2 mM [-32P]ATP, 15 ng of cvRtp1, and Pi, PPi, or PPPi as specified were incubated for 15 min at 37 °C. The extents of ATP hydrolysis in the presence of the phosphate-containing compounds were normalized to the control level of ATP hydrolysis in their absence (defined as 1.0) and then plotted as a function of inhibitor concentration.

The mechanism of inhibition was evaluated by analysis of the effects of increasing PP_i and PPP_i concentrations on ATP hydrolysis at three different concentrations of the [-³²P]ATP substrate (25, 50, and 100 µM ATP). The activity data for each concentration of ATP were transformed into a Dixon plot of 1/v versus the concentration of PP_i or PPP_i (Fig. 8). Both plots revealed a series of lines that converged about a discrete point to the left of the y axis; this pattern is indicative of competitive inhibition (23). The apparent inhibition constants (K_i) were 2.4 µM for pyrophosphate and 0.6 µM for tripolyphosphate (Fig. 8). We reported previously that the K_m of cvRtp1 for ATP is 7 µM (4). These values confirm the inferences from the simple titration curves in Fig. 7 that pyrophosphate and especially tripolyphosphate bind more avidly to the cvRtp1 active site than does the ATP substrate.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Competitive inhibition of cvRtp1 ATPase by tripolyphosphate and pyrophosphate. Reaction mixtures (10 µl) containing 50 mM Tris-HCl, pH 8.0; 5 mM DTT; 1 mM MnCl2; 3 ng of cvRtp1; 25, 50, or 100 µM [-32P]ATP; and varying concentrations of PPi or PPPi were incubated for 15 min at 37 °C. The reaction products were analyzed by TLC. The mechanism of inhibition and the inhibition constants (Ki) were determined from a Dixon plot of the reciprocal of the reaction velocity (nmol 32Pi released/min) versus the concentration of inhibitor (pyrophosphate in A or tripolyphosphate in B) at each fixed concentration of the ATP substrate (25 µM, ; 50 µM, ; or 100 µM, ).

cvRtp1 Has an Intrinsic Metal-dependent Tripolyphosphatase Activity-- The inhibition experiments showed that tripolyphosphate and pyrophosphate bind to the active site of cvRtp1, but they did not address whether these compounds might be substrates for cvRtp1. We employed a colorimetric assay to measure cvRtp1-catalyzed release of inorganic phosphate from unlabeled PPP_i or PP_i in the presence of 1 mM manganese. Reactions containing equivalent concentrations of unlabeled ATP served as a positive control. The extent of P_i release from ATP was proportional to the amount of input cvRtp1 (Fig. 9A). From the slope of the titration curve, we calculated an ATPase turnover number of 1.5 s¹, which agrees with the value determined previously for cvRtp1 using the standard radioisotopic assay of ATP hydrolysis (4). The instructive finding was that cvRtp1 also catalyzed the release of P_i from tripolyphosphate, albeit much less effectively than it hydrolyzed ATP, as indicated from the shift to the right in the titration curve (Fig. 9A). Nonetheless, the experiment showed that the molar yield of P_i at 4 µg of input cvRtp1 was ~80% of the molar amount of input tripolyphosphate, suggesting that the reaction entailed the conversion of PPP_i to P_i and PP_i. The calculated turnover number for the tripolyphosphatase activity of cvRtp1 (0.05 s¹) was 3.3% of its ATPase activity. Consistent with the proposed tripolyphosphatase reaction scheme, we detected no hydrolysis of PP_i to P_i over the same range of enzyme concentrations.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Hydrolysis of tripolyphosphate by cvRtp1. A, enzyme titration. Reaction mixtures (50 µl) containing 50 mM Tris-HCl, pH 8.0; 5 mM DTT; 1 mM MnCl2; 0.2 mM of either PPPi, PPi, or ATP; and cvRtp1 as indicated were incubated for 15 min at 37 °C. The extent of Pi release is plotted as function of input protein. B, divalent cation dependence. Reaction mixtures (50 µl) containing 50 mM Tris-HCl, pH 8.0, 0.2 mM tripolyphosphate, 2 µg of cvRtp1, and MnCl2 or MgCl2 as specified were incubated for 15 min at 37 °C. Phosphate release is plotted as a function of divalent cation concentration.

The novel tripolyphosphatase activity of cvRtp1 was strictly dependent on a divalent cation cofactor (Fig. 9B). Hydrolysis of 0.2 mM tripolyphosphate was optimal at 1-2 mM MnCl₂ and declined slightly at 5-10 mM MnCl₂. Magnesium did not satisfy the metal requirement (Fig. 9B). Thus, the specificity of the tripolyphosphatase for manganese is the same as that of the ATPase activity of cvRtp1 (4). Further evidence that the tripolyphosphatase activity was intrinsic to recombinant cvRtp1 was provided by the finding that the E24A mutant of cvRtp1, which was inert for ATP hydrolysis, was also inert for the hydrolysis of PPP_i (data not shown).

Conclusions and Implications-- The results of the present mutational analysis of Chlorella virus RNA triphosphatase suggest that its active site is located within a hydrophilic tunnel similar to that of S. cerevisiae Cet1 and that the constellation of functional groups responsible for phosphohydrolase reaction chemistry is substantially the same in the Chlorella virus and fungal enzymes. However, there are differences in structure-activity relationships at what we take to be equivalent positions of cvRtp1 and Cet1 that suggest subtle mechanistic distinctions as well as a context-dependent requirement for some of the class III side chains that are proposed to promote catalysis indirectly.

Our results highlight structural differences between cvRtp1 and the RNA triphosphatases encoded by other large DNA viruses. Although the poxvirus, ASFV, and baculovirus RNA triphosphatases do contain the conserved metal-binding glutamates (equivalent to Glu²⁴, Glu²⁶, and Glu¹⁶⁵ of cvRtp1) within two motifs widely separated in the primary structure, they lack obvious counterparts of the other catalytic motifs of cvRtp1 (defined herein and corresponding to the strands of a Cet1-like tunnel). Our inference is that the vaccinia, ASFV, and baculovirus triphosphatases either lack a topologically closed tunnel architecture or, if they retain such a fold, do so without any recognizable amino acid sequence similarity to the Chlorella virus and yeast enzymes. In either case, the implication is that the triphosphatase components of the Chlorella virus, vaccinia virus, and baculovirus capping apparatus have diverged significantly during their evolution. A common origin for poxviruses, ASFV, and Chlorella virus has been proposed based on the hypothesis of a set of shared gene products (predominantly enzymes) that existed in their last common ancestor virus (24). Our mutational analysis showing that the Chlorella virus triphosphatase is closely related to the yeast enzyme is not suggestive of a simple common origin for the respective DNA virus capping systems. Indeed, there are additional major differences in the physical linkage of the enzymatic components of the capping machinery among the large eukaryotic DNA viruses. Chlorella virus encodes separate RNA triphosphatase and guanylyltransferase polypeptides (as do fungi), whereas poxviruses and ASFV each encode a single polypeptide containing triphosphatase, guanylyltransferase, and methyltransferase active sites. We assume, by parsimony, that separately encoded enzymatic domains are the ancestral state and that polyfunctional catalysts of the same metabolic pathway arise via gene fusion events. Polyfunctional capping enzymes related to the poxvirus and ASFV proteins are encoded by cytoplasmic plasmids of fungi (25, 26). Thus, we envision a model whereby the Chlorella virus capping system arose by acquisition of nuclear capping enzymes (likely from a fungus or another lower eukaryote), whereas the polyfunctional capping enzymes of metazoan DNA viruses originated with a cytoplasmic episome (also perhaps fungal) that had already undergone fusion of the capping enzyme genes.

The metal-dependent RNA triphosphatase family to which cvRtp1 and Cet1 belong is recommended as a potential target for antifungal and antiprotozoal drugs that would selectively interfere with the capping of pathogen mRNAs (15, 17, 19). This recommendation is based on the findings that the structure and catalytic mechanism of mammalian RNA triphosphatase are completely different from those of the metal-dependent fungal/protozoal/viral triphosphatases and that mammalian cells encode no homologs of the fungal, protozoal, or DNA virus triphosphatases (19, 27). The high overall similarity in the active sites of cvRtp1 and Cet1 hints that a mechanism-based inhibitor of one triphosphatase family member might display broad spectrum inhibitory activity against other member enzymes. Tripolyphosphate, shown here to be a potent competitive inhibitor of cvRtp1, also inhibits S. pombe triphosphatase Pct1 (14) and S. cerevisiae Cet1.² Thus, tripolyphosphate provides a worthwhile platform on which to build and test new derivatives.

Tripolyphosphate binds more tightly to the cvRtp1 active site than does the ATP substrate. It is likely that tripolyphosphate occupies the site on the enzyme normally filled by the 5' triphosphate component of the NTP or RNA substrate. Our demonstration that cvRtp1 can hydrolyze tripolyphosphate supports this view and provides the first evidence that the nucleoside component of the substrate is not absolutely essential for reaction chemistry. A simple explanation for the higher affinity of cvRtp1 for PPP_i versus ATP is that the former has an extra negative charge on the phosphate occupying the "-phosphate" site in the tunnel that enables an additional electrostatic or hydrogen-bonding contact with the enzyme that is normally not available to the bridging 5'-O of the nucleoside. Transient-state kinetic analysis will be required to determine whether the low tripolyphosphatase activity of cvRtp1 under steady-state conditions is attributable to an inherently slow chemical step or to rate-limiting dissociation of the PP_i product of the tripolyphosphatase reaction.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM42498.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.

To whom correspondence should be addressed: Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. E-mail: s-shuman@ski.mskc.org.

Published, JBC Papers in Press, February 13, 2002, DOI 10.1074/jbc.M200532200

² C. Gong and S. Shuman, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ASFV, African swine fever virus; cvRtp1, Chlorella virus RNA triphosphatase; PP_i, pyrophosphate; PPP_i, tripolyphosphate; DTT, dithiothreitol.

	REFERENCES

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