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J. Biol. Chem., Vol. 281, Issue 28, 18953-18960, July 14, 2006
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1
2
From the
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021
Received for publication, March 27, 2006 , and in revised form, May 12, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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cells. We performed a genetic screen for mutations in the catalytic subunit that bypassed the requirement for the stimulatory subunit in vivo. We thereby identified missense changes in vicinal residues Tyr-752 (to Ser, Cys, or His) and Asn-753 (to Ile), which are located in the cap guanine-binding pocket. Biochemical experiments illuminated a mechanism of intersubunit allostery, whereby the vD12 subunit enhances the affinity of the catalytic subunit for AdoMet and the cap guanine methyl acceptor by 6- and 14-fold, respectively, and increases kcat by a factor of 4. The bypass mutations elicited gains of function in both vD12-independent and vD12-dependent catalysis of cap methylation in vitro when compared with wild-type vD1-(540–844). These results highlight the power of yeast as a surrogate model for the genetic analysis of interacting poxvirus proteins and demonstrate that the activity of an RNA processing enzyme can be augmented through selection and protein engineering. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The architecture of the subunit interface in the poxvirus capping enzyme is undefined, as is the mechanism by which vD12 activates the catalytic subunit. Early studies indicated that vD12 did not affect the extent of UV-induced cross-linking of either AdoMet or GTP to the active site on the vD1-C subunit (19, 20). These findings, and subsequent studies of UV cross-linking to the 5'-guanylate of capped RNA (16), suggested an allosteric effect of vD12 at the active site. Because the vD12 protein is essential for vaccinia replication (21) and because there is no discernible homolog of vD12 in the known proteomes of any eukaryal organism (or of any virus besides the poxviruses), we proposed that vD12 and activated cap methylation are promising targets for antipoxviral drug discovery.
To facilitate genetic and pharmacologic studies of the poxvirus capping enzymes, we have developed yeast-based systems in which cell growth depends on catalysis of cap synthesis by poxvirus proteins (17, 22). We showed that the vaccinia cap methyltransferase domain can function in lieu of the essential yeast enzyme Abd1 and that its in vivo activity requires coexpression of the catalytic vD1-C and stimulatory vD12 subunits (17). The yeast complementation assay has been used as the primary screen to identify individual amino acids of the catalytic and regulatory subunits that are important for cap methylation in vivo (17, 23). For example, a double-alanine scan covering 56 residues of the vD12 subunit identified two lethal mutations and 10 temperature-sensitive alleles. We used this mutant collection to perform a forward genetic screen for second-site suppressors, which defined a constellation of amino acids in vD1 at which mutations restored methyltransferase function in conjunction with defective vD12 proteins (23). Reference to the crystal structure of the microsporidian cap methyltransferase (14) suggested that distinct functional classes of suppressors were selected, including: (i) those that map to surface-exposed loops, which might comprise the physical subunit interface, and (ii) those in or near the substrate-binding sites, which might affect intersubunit allostery. However, none of the suppressors identified in the initial screen were able to bypass the requirement for vD12.
Here, we performed a new genetic screen for vD1-(540–844) mutants that no longer require the stimulatory vD12 subunit to achieve the level of cap methylation required for yeast growth. We recovered bypass suppressor mutations at two vicinal residues, Tyr-752 and Asn-753, that map to the guanosine-binding pocket. We show that vD12 enhances the affinity of the catalytic subunit for the methyl donor and the methyl acceptor and also increases kcat. The bypass mutations stimulated both vD12-independent and vD12-dependent catalysis of cap methylation in vitro when compared with wild-type vD1-(540–844). Our results indicate that bypass suppression occurs via partial mimicry of the intersubunit allostery, and they highlight the plasticity of an RNA processing enzyme under selective pressure.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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120,000 individual ampicillin-resistant bacterial transformants.
Selection of vD1 Mutants That Bypass the Requirement for vD12
Saccharomyces cerevisiae strain YBS40 is deleted at the chromosomal ABD1 locus encoding the yeast cap methyltransferase. Growth of YBS40 depends on the maintenance of plasmid p360A-ABD1(CEN URA3 ADE2 ABD1). abd1 cells were transformed with the 2µ vD1-(540–844) mutant library. Approximately 45,000 Trp+ yeast transformants were replicaplated to agar medium containing 0.75 mg/ml 5-fluoroorotic acid (FOA) and incubated at 25 °C to select for loss of the CEN URA3 ADE2 ABD1 plasmid. The FOA-resistant colonies were replica-plated again to agar medium containing FOA and incubated at 25 °C. Individual FOA-resistant colonies (n = 82) were tested for adenine auxotrophy to ensure loss of the CEN URA3 ADE2 ABD1 plasmid. Ade– abd1 isolates (n = 72) containing candidate bypass mutants (vD1-B alleles) were replica-plated on YPD agar at 19, 25, 30, and 34 °C. Plasmid DNA was isolated from the 19 yeast vD1-B strains that grew at all temperatures tested. The vD1-B plasmids were clonally amplified by transformation in E. coli. Plasmids isolated from single bacterial transformants were retested for suppression by transforming them into abd1 yeast followed by plasmid-shuffle at 25 °C and then gauging growth on YPD agar at 25, 30, 34 and 37 °C (see Fig. 1).
Recombinant Wild-Type and Mutant Catalytic Subunits
The wild-type D1-(540–844) gene and mutant genes Y752S, Y752A, and N753I were PCR-amplified with primers that introduced a BglII site over the start codon and a XhoI site 3' of the stop codon. The PCR products were digested with BglII and XhoI and inserted into the bacterial expression plasmid pET-His10-Smt3. pET-His10Smt3-D1-(540–844) was transformed into E. coli BL21-CodonPlus(DE3). Cultures (500 ml) derived from single transformants were grown at 37 °C in LB medium containing 50 µg/ml kanamycin and 50 µg/ml chloramphenicol until the A600 reached 0.6. The cultures were adjusted to 0.2 mM isopropyl-1-thio-
-D-galactopyranoside and 2% ethanol, and incubation was continued for 20 h at 17 °C. Cells were harvested by centrifugation and stored at –80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol). Phenylmethylsulfonyl fluoride and lysozyme were added to final concentrations of 500 µM and 100 µg/ml, respectively. After incubation on ice for 30 min, Triton X-100 was added to a final concentration of 0.1%, and the lysates were sonicated to reduce viscosity. Insoluble material was removed by centrifugation. The soluble extracts were mixed for 30 min with 1 ml of Ni2+-nitrilotriacetic acid-agarose (Qiagen) that had been equilibrated with buffer A containing 0.01% Triton X-100. The resins were recovered by centrifugation, resuspended in buffer A, and poured into columns. The columns were washed with 10 ml of 20 mM imidazole in buffer A and then eluted stepwise with 1.5-ml aliquots of buffer A containing 50, 100, 250, and 500 mM imidazole. The 250 mM imidazole eluates containing the recombinant catalytic subunits were dialyzed against buffer B (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 2 mM dithiothreitol, 2 mM EDTA, 0.01% Triton X-100) and then stored at –80 °C. The protein concentrations were determined by SDS-PAGE analysis of serial dilutions of the vD1-(540–844) preparations in parallel with serial dilutions of a bovine serum albumin standard. The gels were stained with Coomassie Blue, and the staining intensities of the vD1-(540–844) and bovine serum albumin polypeptides were quantified using a Fujifilm FLA-5000 digital imaging and analysis system. vD1-(540–844) concentrations were calculated by interpolation to the bovine serum albumin standard curve.
Wile-Type and Mutant Methyltransferase Heterodimers
pET-His10Smt3-D1-(540–844) plasmids encoding tagged wild-type and mutant catalytic subunits were transformed into E. coli BL21-CodonPlus(DE3) together with plasmid pET-D12 encoding the nontagged stimulatory subunit. The His10-Smt3-D1-(540–844) and vD12 proteins were produced by isopropyl-1-thio--D-galactopyranoside induction of 500-ml cultures grown in LB medium containing 50 µg/ml kanamycin, 100 µg/ml ampicillin, and 50 µg/ml chloramphenicol. The tagged vD1-C subunits and the associated vD12 subunit were purified from soluble extracts by nickel-agarose chromatography as described above.
Methyltransferase Assay
Reaction mixtures containing 50 mM Tris-HCl, pH 7.5, 5 mM dithiothreitol, GTP, or GpppA as specified, [3H-CH3]AdoMet as specified, and enzyme were incubated for 20 min at 37 °C. Aliquots (4 µl) were spotted on polyethyleneimine-cellulose TLC plates, which were developed with 0.2 M ammonium sulfate (for GTP methyl acceptor) or 0.05 M ammonium sulfate (for GpppA methyl acceptor). The AdoMet- and m7GTP- or m7GpppA-containing portions of the lanes were cut out, and the radioactivity in each was quantified by liquid scintillation counting.
Kinetic Parameters
Methyl Acceptor Titrations—Reaction mixtures (20 µl) contained 50 µM [3H-CH3]AdoMet, increasing concentrations of GTP or GpppA, and wild-type or mutant vD1-C catalytic subunits or wild-type and mutant vD1-C/vD12 heterodimers.
AdoMet Titrations—Reaction mixtures (20 µl) contained 5 mM GTP, increasing concentrations of [3H-CH3]AdoMet, and wild-type or mutant vD1-C catalytic subunit or wild-type and mutant vD1-C/vD12 heterodimers. The extents of methyl transfer were plotted as a function of the variable substrate concentration. Km and kcat values were calculated from double-reciprocal plots of the data. The results are summarized in Table 2. Each datum is the average of three independent substrate titration experiments (± mean error).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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strain bearing ABD1 on a URA3 plasmid. After selection for growth on medium containing FOA to eliminate ABD1, we recovered 19 strains that contained putative D12 bypass alleles of vD1-(540–844), which were named vD1-B1, vD1-B2, etc. After clonal amplification in bacteria, the 2µ vD1-B plasmids were retransformed into abd1. Although the wild-type vD1-(540–844) gene was unable to sustain growth on FOA, each of the vD1-B alleles supported growth on FOA. The 19 plasmids that retested positive for bypass were then sequenced to identify the coding changes associated with the bypass phenotype (Table 1).
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The Y752S and N753I strains grew as well as wild-type vD1-C vD12 cells at 25, 30, and 34 °C (as gauged by colony size) but failed to grow at 37 °C (Fig. 1). The Y752C strain grew at 25 and 30 °C but was barely viable at 34 °C and failed to grow at 37 °C. Another bypass suppressor containing two missense changes, Y752H and Y668H, grew at 25, 30, and 34 °C but not at 37 °C. Although each of the bypass mutations conferred a temperature-sensitive phenotype in the absence of vD12 (Fig. 1), none of the vD1-B alleles displayed a growth defect in the presence of vD12 (data not shown), suggesting that the bypass mutations in the catalytic subunit did not adversely affect the physical or functional interactions of the methyltransferase subunits in vivo.
The vD1-B alleles were transferred to CEN plasmids and tested for abd1 complementation. Each allele in single copy sustained cell growth in the absence of vD12 (Fig. 2). Again, Y752S and N753I cells displayed the best growth when compared with wild-type vD1-C vD12 cells (Fig. 2); however, Y752S and N753I failed to grow at 34 °C (not shown). Thus, decreasing the gene copy number lowered their restrictive growth temperatures in the absence of vD12 from 37 to 34 °C. The restrictive temperatures for CEN Y752H/Y668H and Y752C strains were also lowered when compared with the strains carrying these genes on 2µ plasmids (Figs. 1 and 2).
Additional insight to the basis for suppression by mutations at Tyr-752 was obtained by testing two purposefully constructed vD1-(540–844) alleles, Y752A and Y752F, which either removed the entire side chain beyond the -carbon or eliminated only the hydroxyl while maintaining the aromatic character of the side chain. Although the single-copy Y752A allele bypassed the need for vD12 (Fig. 2), Y752F did not (data not shown; note that the Y752F allele is fully functional in yeast in the presence of vD12). The Y752A strain grew as well as Y752S at 19, 25, and 30 °C but failed to grow at 34 or 37 °C (Fig. 2).
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is not attributable to gross stabilization of the catalytic subunit against proteolytic decay. Second, whereas the Y752S and N753I mutants of vD1-C accumulated to higher levels that the wild-type subunit in the absence of vD12, their relative levels were not further increased when D12 was present. At least some of the apparent increase in abundance of the N753I, Y758H/Y668H, and Y752C subunits in cells coexpressing vD12 (relative to wild-type vD1-C) correlated with increased amounts of protein applied to the lanes (as judged by the increased level of Prp43) (Fig. 3). The immunoblotting results suggest that bypass of the vD12 requirement by point mutations was not solely a matter of increasing the amount of vD1-C but rather involved, at least in part, an effect on the methyltransferase activity of the mutant subunits. An analysis of recombinant versions of the vD12-independent vD1-C subunits was informative in this regard, as described below. Biochemical Basis for Activation of vD1-C by vD12—To probe the mechanism of activation of vD1-(540–844) by vD12, we evaluated the kinetic parameters of the methylation reaction catalyzed by vD1-(540–844) alone versus the reaction of the vD1-(540–844)/vD12 heterodimer. The catalytic subunit was produced in E. coli as a His10-Smt3 fusion protein and purified from a soluble bacterial lysate by nickel-agarose chromatography (Fig. 4). The recombinant methyltransferase heterodimer was produced by coexpressing His10-Smt3-D1-(540–844) and untagged vD12. As reported previously (6), vD12 formed a heterodimer with tagged vD1-C in E. coli, which could be recovered from a soluble bacterial extract by nickel-agarose chromatography (Fig. 4). To assay methyltransferase activity, we used GTP as the methyl acceptor and [3H-CH3]AdoMet as the methyl donor. The reaction products were separated by PEI-cellulose TLC, and the transfer of the tritiated methyl group to generate labeled m7GTP was quantified. The assays were conducted at 30 °C in light of our initial findings that the specific activity of D1-(540–844) was 2.8-fold higher at 30 °C than at 37 °C, whereas the D1-(540–844)/D12 heterodimer was equally active at 30 °C and 37 °C (data not shown).
Kinetic parameters were determined by titrating GTP at a fixed concentration of AdoMet (50 µM) and titrating AdoMet at a fixed concentration of GTP (5 mM). The results are summarized in Table 2. The catalytic subunit on its own had apparent Km values of 420 µM for GTP and 20 µM for AdoMet, with a kcat of 0.4 min–1. The heterodimeric enzyme had Km values of 30 µM GTP and 3.2 µM AdoMet, with a kcat of 1.7 min–1. Thus, D12 increased the affinity for GTP by 14-fold and for AdoMet by a factor of 6 while increasing kcat by a factor of 4. If one estimates "catalytic efficiency" as kcat/(KmAdoMet x KmGTP), then the D12 subunit enhances catalytic efficiency by a factor of 370. It is worth noting that the apparent affinities of the heterodimeric methyltransferase for AdoMet and GTP determined here are in excellent agreement with the Km values of 3 µM AdoMet and 36 µM GTP reported previously by Higman et al. (5). However, there are no prior kinetic data for the methyltransferase reaction of the catalytic domain by itself.
We also assayed methyltransferase activity using the cap dinucleotide GpppA as the methyl acceptor. From GpppA titration experiments at 50 µM AdoMet, we determined apparent Km values of 639 and 62 µM GpppA for the catalytic subunit and the heterodimeric enzyme, respectively (Table 2). The 10-fold increase in affinity for the GpppA methyl acceptor elicited by the vD12 subunit was consistent with the similar increase in affinity for GTP described above. The kcat values with GpppA were 1.3 and 2.1 min–1 for vD1-C and vD1-C/vD12, respectively (Table 2). These experiments provide a coherent biochemical explanation for the allosteric stimulation of methyltransferase activity by the vD12 subunit, which is dominated by increased affinity for substrates, especially the methyl acceptor, and also entails an increased turnover number.
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Kinetic analysis of the mutant heterodimers revealed that the Y752A, Y752S, and N753I subunits remained fully responsive to the allosteric effects of vD12 on substrate affinity and turnover number, indeed even more so than the wild-type catalytic subunit. For example, the Km values for AdoMet of the mutant heterodimers (2.2 µM for Y752A, 1.8 µM for Y752S, and 1.4 µM for N753I) were lower by 15-, 14-, and 27-fold when compared with the isolated mutant catalytic subunits and were even slightly lower than the Km value of 3.2 µM AdoMet seen for the wild-type heterodimer (Table 2). Similarly, the Km values for GTP of the mutant heterodimers (14 µM for Y752A, 19 µM for Y752S, and 5.6 µM for N753I) were lower by 22-, 18-, and 47-fold when compared with the isolated mutant catalytic subunits and were also lower than the Km value of 30 µM GTP determined for the wild-type heterodimer (Table 2). The kcat values of the mutant heterodimers for GTP methylation (7.1 min–1 for Y752A, 5.7 min–1 for Y752S, and 19 min–1 for N753I) were all significantly higher than the turnover number for the wild-type vD1-C/vD12 methyltransferase. Indeed, the calculated catalytic efficiency of the N753I/vD12 enzyme was 140-fold greater than that of the wild-type vD1-C/vD12 enzyme.
Similar gains of function were observed for methylation of GpppA, whereby the Km values for GpppA of the mutant heterodimers (17 µM for Y752A, 21 µM for Y752S, and 7.1 µM for N753I) were lower by 26-, 27-, and 66-fold when compared with the isolated mutant catalytic subunits and were significantly lower than the Km value of 62 µM GpppA determined for the wild-type heterodimer (Table 2). The kcat values of the mutant heterodimers for GpppA methylation (11 min–1 for Y752A, 8.3 min–1 for Y752S, and 17 min–1 for N753I) were 4–8-fold higher than the turnover number of 2.1 min–1 for the wild-type vD1-C/vD12 methyltransferase. The catalytic efficiency of the N753I/vD12 enzyme in GpppA methylation was 160-fold greater than that of the wild-type vD1-C/vD12 enzyme.
vD12 Stabilizes vD1-C against Thermal Inactivation—Although the gains of activity of the isolated catalytic subunits elicited by Tyr-752 and Asn-753 mutations provide a satisfying explanation for bypass of the vD12 requirement in vivo, we conducted additional experiments to illuminate why the vD1-B yeast strains were consistently temperature-sensitive for growth. We tested the thermal stability of the wild-type and mutant vD1-C proteins, as isolated subunits and as heterodimers with vD12. The proteins were preincubated at increasing temperatures, from 30 to 50 °C in 5 °C increments, and then quenched immediately on ice. Aliquots of the protein samples were assayed for GTP methylation at 30 °C. The decay of methyltransferase activity as a function of preincubation temperature is plotted in Fig. 5A. The methyltransferase activity at each pretreatment temperature was normalized to the activity of the untreated control sample of the same enzyme preparation (defined as 1.0). The results showed that the isolated Y752A, Y752S, and N753I catalytic subunits were heat-labile when compared with the wild-type vD1-(540–844) subunit, e.g. the mutants retained 24–28% of their activity after preincubation at 30 °C and 1–6% of their activity after treatment at 40 °C, whereas the wild-type subunit retained 90 and 35% of its activity after the same preincubations. The vD12 subunit exerted a stabilizing effect on the wild-type catalytic subunit, seen as an 5°C shift to the right in the thermal inactivation curve (Fig. 5A). The stabilizing effect of vD12 was even more pronounced for the Y752A, Y752S, and N753I mutants, which were restored to near wild-type thermal sensitivity in the context of the heterodimeric enzyme (Fig. 5A). These in vitro results explain why the ts phenotype of the yeast vD1-C strains is eliminated by vD12 coexpression in vivo. The enhanced sensitivity of the isolated mutant catalytic subunits during preheating (in the absence of substrates) raised the issue of whether the enzymes were prone to inactivation during the methyltransferase reactions performed at 30 °C. An analysis of the time course of methylation by the wild-type and mutant vD1-C catalytic subunits, using enzyme concentrations adjusted to attain similar levels of activity in each reaction, indicated that this was not the case (Fig. 5B).
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DISCUSSION |
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Reference to the crystal structure of the cellular cap methyltransferase Ecm1 (14) suggests that Tyr-752 and Asn-753 are located in the cap guanine-binding pocket. The residue corresponding to Tyr-752 is conserved as an aromatic side chain in Ecm1 and other cellular cap methyltransferases. Bypass of vD12 occurred when Tyr-752 was changed to serine, alanine, or cysteine (and also to histidine in the context of a double mutation with Y668H) but not when replaced by phenylalanine. These results suggest that the gain-of-function is attributable to a reduction in the size of the side chain. The equivalent residue in Ecm1 is Phe-214, which lines the cap guanosine-binding pocket and makes van der Waals contacts to the N2 and N3 atoms of the guanine base via C
2 and C
2 of the phenyl ring (14). The position of the guanine in the pocket of Ecm1-GTP cocrystal was suggested to approximate that of the cap acceptor prior to methyl transfer, yet there was a clash between the guanine-N7 atom in the Ecm1-GTP complex and the AdoMet methyl group in the Ecm1-AdoMet complex when the two complexes were superimposed. Thus, it was proposed that the position of the cap guanosine must shift in the bisubstrate complex order to attain the proper orientation of the reactive moieties (14). This idea is consistent with UV cross-linking studies of the heterodimeric vaccinia cap methyltransferase, which showed that cross-linking of vD1-C to the cap guanylate is strongly stimulated by simultaneous occupancy of the methyl donor site (16). We envision that the allosteric activation of the poxvirus catalytic subunit by the stimulatory vD12 subunit entails a conformational change in the guanosine-binding pocket that accommodates the cap in a catalytically productive geometry, in which case the circumvention of its reliance on the stimulatory subunit can be achieved by downsizing Tyr-752 to allow greater freedom of movement of the cap guanine.
Asn-753 is conspicuously not conserved in Ecm1 or most other cellular cap methyltransferases. This position is occupied by threonine in Ecm1, tryptophan in Saccharomyces and Candida cap methyltransferases, tyrosine in Schizosaccharomyces cap methyltransferase, and asparagine in the human enzyme. However, Asn-753 is conserved as asparagine in the D1 homologs of the vast majority of vertebrate poxviruses for which genome sequence is available. The equivalent residue in Ecm1 is Thr-215, which, although part of a strand that forms the cap-binding pocket, is oriented away from the cap guanosine so that the threonine side chain is exposed on the protein surface (14). We presume that the N753I change, which affords the greatest gain of catalytic function of the mutations described here, elicits its effect via a subtle conformational change in the substrate-binding pocket that partially mimics the action of the vD12 subunit.
By analyzing the kinetic parameters of the isolated wild-type catalytic subunit and comparing them with that of the wild-type heterodimeric methyltransferase, we obtained evidence for a mechanism of allosteric activation entailing enhanced affinity for the AdoMet methyl donor and the guanosine methyl acceptor (either GTP or GpppA) plus increased turnover number. From the Km values for AdoMet and GTP and kcat values, we estimated that the D12 subunit increases catalytic efficiency by a factor of 370. Although earlier studies had shown that the yield of UV cross-linked photoadduct formation between vD1-C and either [3H]AdoMet or [32P]GTP was similar for the isolated catalytic subunit and the methyltransferase heterodimer (19, 20), it must be kept in mind that cross-linking, although of great value in establishing that the substrate-binding sites reside within the D1 subunit, does not directly address the affinity of the enzyme for catalytically productive binding of the substrates. This is because the cross-linking efficiency depends on both binding of the substrate and its proximity to a suitable reactive moiety on the enzyme.
The cross-linking of the vD1-C/vD12 heterodimer to [3H]AdoMet occurred with high efficiency (40% of the input D1 protein being photolabeled), and the observed half-saturation of cross-linking at 3 µM AdoMet (19) agrees with the Km for AdoMet reported here and previously (5). The amount of AdoMet photoadduct to the isolated catalytic subunit in the presence of 20 µM AdoMet was about half that seen with the native methyltransferase homodimer (19), which is consistent with our present finding that the Km of the isolated catalytic subunit for AdoMet is 20 µM. In the earlier studies of GTP photocross-linking, which were performed in the absence of AdoMet, the efficiency of photocross-linking was low (<1% of the input enzyme was photolabeled), but the observed half-saturation of cross-linking to the heterodimeric enzyme at 35 µM (20) agrees with the Km values for GTP reported here and previously. Although it was noted that adding vD12 to the catalytic subunit resulted in a 2-fold increase in cross-linking to 10 µM GTP (20), the authors surmised that activation of cap methylation by vD12 was not achieved by enhancement of substrate binding but principally through a conformational change of the active site that makes it more catalytically active. Our determinations of kcat support the second part of the their conclusion, but the Km data vitiate their inferences about the contributions of substrate affinity. Observations that the UV cross-linking of the cap guanylate methyl acceptor to the catalytic subunit is stimulated by simultaneous binding of the methyl donor (16) reinforce the concept that measurements of Km for the methyl acceptor in the presence of AdoMet provide the better and more direct assay of productive substrate affinity than does GTP cross-linking in the absence of AdoMet. Indeed, the large increase in affinity for GTP or GpppA in the presence of vD12 appears to be a dominant contribution to the overall activation.
The mutations in the catalytic subunit isolated during genetic selection for D12-independent cell growth elicited true gains of catalytic activity in vitro when compared with the wild-type version of vD1-C. The gain of function was evident in the absence and the presence of vD12. Although the mutant catalytic subunits alone gained activity primarily via an increase in kcat, the mutants were more responsive than the wild-type subunit to allosteric activation by vD12, resulting in lower Km for both substrates and increased turnover number. These results show that the native poxvirus guanine-N7 methyltransferase heterodimer is underpowered and not performing to its full potential. This was not a foregone conclusion. Indeed, one can easily imagine that bypass mutations would raise the basal level of activity of the isolated catalytic subunit (which is clearly underpowered, otherwise it would not require a stimulatory subunit) yet not affect the catalytic efficiency in the presence of vD12.
In summary, we demonstrate here that a genetic analysis of interacting poxvirus proteins can be executed to good effect using yeast as a surrogate system. Although yeast had been exploited previously for a two-hybrid interactions analysis of virtually all pairs of poxvirus proteins (24), that analysis did not identify all of the known interaction partners (e.g. D1 and D12 were missed) and did not incorporate a functionally relevant phenotype for any of the viral proteins scrutinized. Also, we show that an essential RNA processing enzyme can evolve improved catalytic efficacy under selective pressure in vivo, via missense mutations at or near the active site. One wonders how many other RNA modifying enzymes might also not be operating at their full catalytic potential.
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1 To whom correspondence may be addressed. E-mail: bschwer{at}med.cornell.edu. 2 An American Cancer Society Research Professor. To whom correspondence may be addressed. E-mail: s-shuman{at}ski.mskcc.org.
4 The abbreviations used are: AdoMet, S-adenosylmethionine; FOA, 5-fluoroorotic acid.
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