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• W1970255065 abstract "The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540–844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1Δ 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. The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540–844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1Δ 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. The 5′-m7GpppN structure of vaccinia virus mRNAs is formed by a virus-encoded two-subunit “capping enzyme” with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7)-methyltransferase activities (1Martin S.A. Moss B. J. Biol. Chem. 1975; 250: 9330-9335Abstract Full Text PDF PubMed Google Scholar, 2Venkatesan S. Gershowitz A. Moss B. J. Biol. Chem. 1980; 255: 903-908Abstract Full Text PDF PubMed Google Scholar). The methyltransferase domain consists of a heterodimer of the carboxyl-terminal portion of the vD1 subunit (vD1-C, spanning amino acids 540–844) and the 287-amino acid polypeptide encoded by the vaccinia D12 gene (3Cong P. Shuman S. J. Biol. Chem. 1992; 267: 16424-16429Abstract Full Text PDF PubMed Google Scholar, 4Higman M.A. Bourgeois N. Niles E.G. J. Biol. Chem. 1992; 267: 16430-16437Abstract Full Text PDF PubMed Google Scholar). The active site is located within vD1-C, which has a weak intrinsic methyltransferase activity that is stimulated by the vD12 subunit (5Higman M.A. Christen L.A. Niles E.G. J. Biol. Chem. 1994; 269: 14974-14981Abstract Full Text PDF PubMed Google Scholar, 6Mao X. Shuman S. J. Biol. Chem. 1994; 269: 24472-24479Abstract Full Text PDF PubMed Google Scholar). The requirement for a stimulatory subunit is what distinguishes the poxvirus cap methyltransferase from cellular cap-methylating enzymes. The latter are monomeric polypeptides that display amino acid sequence similarity to the vaccinia D1 catalytic subunit but not to vD12 (7Mao X. Schwer B. Shuman S. Mol. Cell. Biol. 1995; 15: 4167-4174Crossref PubMed Scopus (91) Google Scholar, 8Mao X. Schwer B. Shuman S. Mol. Cell. Biol. 1996; 16: 475-480Crossref PubMed Scopus (60) Google Scholar, 9Saha N. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 16553-16562Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Hausmann S. Vivarès C.P. Shuman S. J. Biol. Chem. 2002; 277: 96-103Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Mutational analyses have pinpointed essential side chains conserved in cellular cap methyltransferases and poxvirus D1 proteins (8Mao X. Schwer B. Shuman S. Mol. Cell. Biol. 1996; 16: 475-480Crossref PubMed Scopus (60) Google Scholar, 9Saha N. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 16553-16562Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Hausmann S. Vivarès C.P. Shuman S. J. Biol. Chem. 2002; 277: 96-103Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 11Wang S.P. Shuman S. J. Biol. Chem. 1997; 272: 14683-14689Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Yamada-Okabe T. Mio T. Kashima Y. Matsui M. Arisawa M. Yamada-Okabe H. Microbiology (N. Y.). 1999; 145: 3023-3033Crossref PubMed Scopus (12) Google Scholar, 13Schwer B. Saha N. Mao X. Chen H.W. Shuman S. Genetics. 2000; 155: 1561-1576Crossref PubMed Google Scholar, 14Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 15Hausmann S. Zhang S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Mao X. Shuman S. Biochemistry. 1996; 35: 6900-6910Crossref PubMed Scopus (32) Google Scholar, 17Saha N. Shuman S. Schwer B. J. Virol. 2003; 77: 7300-7307Crossref PubMed Scopus (21) Google Scholar, 18Hall M.P. Ho C.K. RNA (Cold Spring Harbor). 2006; 12: 488-497Google Scholar). The crystal structure of the Encephalitozoon cuniculi cap methyltransferase Ecm1 (14Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) revealed that the amino acid side chains that contact AdoMet 4The abbreviations used are: AdoMet, S-adenosylmethionine; FOA, 5-fluoroorotic acid. 4The abbreviations used are: AdoMet, S-adenosylmethionine; FOA, 5-fluoroorotic acid. and the GTP cap acceptor are conserved in the catalytic subunit of the poxvirus enzyme. 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 (19Higman M.A. Niles E.G. J. Biol. Chem. 1994; 269: 14982-14987Abstract Full Text PDF PubMed Google Scholar, 20Niles E.G. Christen L. Higman M.A. Biochemistry. 1994; 33: 9898-9903Crossref PubMed Scopus (13) Google Scholar). These findings, and subsequent studies of UV cross-linking to the 5′-guanylate of capped RNA (16Mao X. Shuman S. Biochemistry. 1996; 35: 6900-6910Crossref PubMed Scopus (32) Google Scholar), suggested an allosteric effect of vD12 at the active site. Because the vD12 protein is essential for vaccinia replication (21Carpenter M.S. DeLange A.M. J. Virol. 1991; 65: 4042-4050Crossref PubMed Google Scholar) 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 (17Saha N. Shuman S. Schwer B. J. Virol. 2003; 77: 7300-7307Crossref PubMed Scopus (21) Google Scholar, 22Ho C.K. Martins A. Shuman S. J. Virol. 2000; 74: 5486-5494Crossref PubMed Scopus (26) Google Scholar). 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 (17Saha N. Shuman S. Schwer B. J. Virol. 2003; 77: 7300-7307Crossref PubMed Scopus (21) Google Scholar). 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 (17Saha N. Shuman S. Schwer B. J. Virol. 2003; 77: 7300-7307Crossref PubMed Scopus (21) Google Scholar, 23Schwer B. Shuman S. Virology. 2006; (in press)PubMed Google Scholar). 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 (23Schwer B. Shuman S. Virology. 2006; (in press)PubMed Google Scholar). Reference to the crystal structure of the microsporidian cap methyltransferase (14Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) 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. The vD1-(540–844) gene fragment was PCR-amplified in vitro with Taq DNA polymerase. dATP was included as one-fourth of the concentration of the other dNTPs to enhance the frequency of deoxynucleotide misincorporation. The vD1-(540–844) PCR products from four separate amplification reactions (containing 0.05 or 0.1 mm dATP, with or without 0.1 mm MnCl2) were pooled, digested with EcoRI and XhoI endonucleases, and ligated into pYX232 (2μ TRP1). Ligation mixtures were transformed into Escherichia coli by electroporation, and a 2μ plasmid DNA library was isolated from a pool of ∼120,000 individual ampicillin-resistant bacterial transformants. 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). abd1Δ 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– abd1Δ 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 abd1Δ 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). 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-β-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. 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-β-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. 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. 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).TABLE 2Kinetic evidence for intersubunit allostery and gain-of-function effects of bypass mutationsAdoMetGTPGpppAKmkcatKmkcatKmkcatμmmin-1μmmin-1μmmin-1D1-C20 ± 20.4 ± 0.02420 ± 830.4 ± 0.07639 ± 981.3 ± 0.1Y752A32 ± 132.3 ± 0.5309 ± 401.7 ± 0.11442 ± 216.4 ± 0.3Y752S26 ± 71.6 ± 0.3338 ± 141.4 ± 0.1582 ± 526.1 ± 0.4N753I38 ± 125.2 ± 0.7264 ± 583.2 ± 0.42472 ± 156.3 ± 0.3D1-C/D123.2 ± 11.7 ± 0.230 ± 31.4 ± 0.262 ± 92.1 ± 0.2Y752A/D122.2 ± 16.1 ± 0.514 ± 3.67.1 ± 117 ± 3.111 ± 1.6Y752S/D121.8 ± 0.54.9 ± 0.319 ± 4.35.7 ± 0.521 ± 28.3 ± 0.9N753I/D121.4 ± 0.514 ± 0.45.6 ± 0.619 ± 17.1 ± 1.517 ± 0.2 Open table in a new tab Selection of vD1 Mutants That Bypass the Requirement for vD12—To execute a screen for mutations that subvert the requirement for a stimulatory subunit, we PCR-amplified the vD1-(540–844) gene encoding the minimized catalytic subunit under conditions favoring nucleotide misincorporation and cloned the mutated DNA into a multicopy yeast 2μ plasmid. The 2μ vD1-(540–844) mutant library was transformed without a vD12 gene into a yeast abd1Δ 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 abd1Δ. 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).TABLE 1Mutant alleles of vD1 (540–844) that bypass the requirement for vD12vD1-B alleleMutation(s)1, 13, 16Y752S12Y752S, K719R, T811A15, 19, 10Y752C14Y752C, 1727R17Y752H, Y668H8Y752H, M579T, 1613T, A691T, N731D, N776S2, 3, 4, 5, 6, 7, 9, 11, 18N753I Open table in a new tab The bypass screen identified single missense changes in vicinal residues Tyr-752 (to Ser or Cys) and Asn-753 (to Ile) that resulted in vD12-independent growth (Table 1). The single N753I change was recovered nine times; these bypass clones represent at least three independently selected isolates, distinguished by the presence or absence of certain translationally silent mutations within the vD1-C gene. The three bypass clones containing the single Y752S change represent at least two independently selected mutations, and the three Y752C alleles comprise three independently selected bypass suppressors, as judged by the occurrence or absence of unique silent mutations. 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 abd1Δ 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 β-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). The apparent gain of function in vivo conferred by the Tyr-752 and Asn-753 mutants at 25–30 °C could reflect either enhanced stability or enhanced activity of the catalytic subunit in the absence of vD12 or both. We exploited an antibody to vD1-C (3Cong P. Shuman S. J. Biol. Chem. 1992; 267: 16424-16429Abstract Full Text PDF PubMed Google Scholar) to gauge the relative steady-state levels of the catalytic subunit in ABD1 strains expressing wild-type vD1-(540–844) and the various bypass mutants in the absence or presence of coexpressed vD12. Note that it was necessary to express the poxvirus proteins in an ABD1 strain so that the levels of wild-type vD1-C could be evaluated when the vD12 subunit was not available. Whole cell lysates derived from ABD1 strains in log phase growth at 30 °C in selective media for the plasmids bearing vD1-C and vD12 were resolved by SDS-PAGE in parallel with recombinant vD1-C protein; the polypeptides were transferred to membranes and probed by Western blotting. The antibody recognized the recombinant vD1-(540–844) polypeptide and the poxvirus proteins in extracts of yeast strains carrying vD1-C on 2μ plasmids, but no signal was detectable in the control strains carrying the vector plasmid without a vD1-C gene (Fig. 3). Probing the blot with antibody to an endogenous yeast protein, the pre-mRNA splicing factor, Prp43, verified that similar amounts of extract from the vector-containing and vD1-C strains had been loaded. The first finding of note was that the level of wild-type vD1-C in yeast (relative to the recombinant polypeptide standard and the Prp43 loading control) was similar whether or not vD12 was coexpressed, suggesting that the requirement for vD12 for complementation of abd1Δ 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 (6Mao X. Shuman S. J. Biol. Chem. 1994; 269: 24472-24479Abstract Full Text PDF PubMed Google Scholar), 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 × 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. (5Higman M.A. Christen L.A. Niles E.G. J. Biol. Chem. 1994; 269: 14974-14981Abstract Full Text PDF PubMed Google Scholar). 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. Biochemical Basis for Bypass of the vD12 Requirement—To investigate how mutations in the catalytic subunit bypass the requirement for vD12 in vivo, we produced and purified the vD1-C mutants Y752A, Y752S and N753I as isolated catalytic subunits and as heterodimers with vD12 (Fig. 4) and determined their kinetic parameters for AdoMet-dependent methylation of GTP and GpppA (Table 2). The apparent kcat values for methylation of 5 mm GTP by the isolated Y752A, Y752S, and N753I mutants at saturating AdoMet were 2.3, 1.6, and 5.2 min–1, respectively, which represent increases of 6-, 4-, and 13-fold when compared with kcat for the isolated wild-type catalytic subunit. The kcat values for methylation of GpppA by Y752A, Y752S, and N753I were 6.4, 6.1, and 6.3 min–1, respectively, which were about 5-fold higher than the kcat of wild-type D1-(540–844). Moreover, the turnover numbers of the Y752A, Y752S, and N753I subunits in methylation of GTP or GpppA equaled or exceeded the turnover numbers of the wild-type methyltransferase heterodimer (Table 2). These results signify that bypass reflects a true gain of function in catalysis for the vD1-C subunit. This gain of activity of the mutant catalytic subunits was not attributable to increased affinity for AdoMet (where values ranged from 26 to 38 μm) and entailed, at best, a modest (less than 2-fold) increase in affinity for the methyl acceptor. Thus, the mutations that bypass the vD12 requirement in vivo mimic only partially the allosteric effects of vD12 on the vD1-C activity in vitro. 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 th" @default.
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