Pyridoxal Phosphate Inhibits Dynamic Subunit Interchange among Serine Hydroxymethyltransferase Tetramers*

Krista A. Zanetti and Patrick J. StoverDagger

From the Cornell University, Division of Nutritional Sciences, Ithaca, New York 14853

Received for publication, November 13, 2002, and in revised form, January 2, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT)1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate (1, 2). This reaction is a primary source of single carbons that are required for cytoplasmic one-carbon metabolism. SHMT is present in the cytoplasm (cSHMT) and mitochondria of eukaryotic cells, and different genes encode the two SHMT proteins (3-5). Both SHMT isozymes are sources of one-carbon units for cytoplasmic one-carbon metabolism (1, 3). In the cytoplasm, folate-activated one-carbon units are required for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine (1, 6). Mitochondrial one-carbon metabolism is necessary for the conversion of serine to formate (7), and the mitochondrial SHMT enzyme catalyzes the first step in this pathway by converting serine and THF to glycine and methylenetetrahydrofolate (6, 7). Formate enters the cytoplasm, where it is incorporated into the folate one-carbon pool (1, 3, 4). The mitochondrial SHMT gene is expressed at similar levels in most mammalian tissues, whereas the cSHMT gene exhibits a dynamic range of tissue-specific expression (5).

Mammalian SHMT enzymes are 55-kDa homotetramers with four active sites/tetramer. High resolution structures are available for the human, mouse, and rabbit cSHMT enzymes, some with amino acid and folate substrates bound (8-10). All of the solved cSHMT structures reveal that the enzyme is best described as a dimer of tight, obligate dimers. Each obligate dimer contains two active sites, and catalytically essential amino acid residues from each monomer contribute to both active sites. Tetramer formation results from the relatively weak association of two obligate dimers. Analysis of the mouse cSHMT structure reveals that the tetramer contact surface is small, involving residues 135-137, 154-157, 168-171, and 189-194 of each monomer (9). Prokaryotic SHMT enzymes lack residues that lie at the tetramer interface and are catalytically active as obligate dimers in solution (11, 12). Mammalian SHMT isozymes are tetramers in solution but form mixtures of dimers and tetramers in the absence of bound PLP (13). The dissociation of cSHMT tetramers into obligate dimers in the absence of bound PLP suggests that the catalytic site of the enzyme communicates with amino residues at the tetramer interface. Site-directed mutations that alter amino acid residues near the tetramer interface site or that decrease the affinity of PLP for the enzyme weaken the interactions between two obligate dimers (13, 14). For example, recombinant D89N cSHMT from sheep has decreased catalytic activity and is a mixture of dimers and tetramers in solution (14). Recombinant H134N cSHMT from sheep has decreased affinity for PLP and is present in solution as a mixture of tetramers and dimers (13). The H134N cSHMT dimers are active, but the specific activity of the enzyme reduced by 75% compared with the nonmutated enzyme under conditions of saturating PLP. This study indicates that tetramer formation is not necessary for cSHMT catalytic activity (13).

To better understand the stability of cSHMT tetramers and the interaction of subunits within cSHMT tetramers and to further examine the relationship between tetramer formation and cSHMT activity, we engineered a catalytically inactive, dominant-negative cSHMT enzyme (DNcSHMT). The results from these studies provide evidence that neither monomers nor obligate dimers exchange among preformed cSHMT tetramers in the presence of PLP. However, loss of cSHMT-bound PLP permits exchange of obligate dimers, but not monomers, among preformed cSHMT tetramers. Furthermore, we show that cSHMT and DNcSHMT heterodimers are catalytically inactive, indicating that the DNcSHMT monomer effectively deactivates endogenous cSHMT activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 5-FormylTHF, allothreonine, alcohol dehydrogenase, NADH, isopropyl beta -D-thiogalactopyranoside, lysozyme, and pyridoxal 5-phosphate were obtained from Sigma. All other chemicals were reagent grade. The restriction enzymes were obtained from Promega and Invitrogen. The pET22b and pET28a vectors were obtained from Novagen. TOPO vector, TOP 10 competent cells, and BL21* competent cells were obtained from Invitrogen.

Generation and Expression of the DNcSHMT cDNA-- The DNcSHMT cDNA was constructed using the human cSHMT cDNA as a template. Site mutations were incorporated into the cSHMT cDNA using the following primers. The forward primer was 5'-tctgagggtacccgggccagagagcctttggcgggactgag-3' with the KpnI site underlined and the altered nucleotides in bold type, which result in Y83A and Y82F codon substitutions in the recombinant protein. The reverse primer was 5'-ttgggatccacacttttcactcctttcctgtagaagatcatgccagctcggcagcctcgcagggtctggtgagtgg-3' with the BamHI site underlined and the altered nucleotides shown in bold type resulting in a K257Q codon substitution in the human protein. The region of the cSHMT cDNA that encodes the targeted amino acid residues was amplified by PCR: 30 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 90 s with a 10-min extension at 72 °C. The DNcSHMT cDNA was generated by replacing the KpnI-BamHI fragment within the human cSHMT cDNA with the PCR product that contains the three codon substitutions. The DNcSHMT cDNA was subcloned into the NdeI and NotI restriction sites of the pET22b expression vector, which confers kanamycin resistance. The cSHMT cDNA was subcloned into pET28a expression vector in frame with the N terminus polyhistidine tag using the NdeI and NotI restriction sites, and this vector confers ampicillin resistance. The mutated cDNAs were sequence-verified. The expression vectors containing the cSHMT and DNcSHMT cDNAs were transformed into competent BL21* bacteria both singly and in combination. One-liter cultures of BL21* cells expressing the cSHMT and DNcSHMT or coexpressing cSHMT and DNcSHMT cDNAs were grown to mid-log phase, and protein synthesis was induced with isopropyl beta -D-thiogalactopyranoside for 8 h at room temperature. The cell pellets were harvested and stored at -80 °C until purification.

Purification of the cSHMT and DNcSHMT Proteins-- The cell pellets were lysed in a buffer containing 40 mM potassium phosphate, pH 7.0, 10 mM 2-mercaptoethanol, and 100 nM PLP using a French press, and the insoluble material was removed by centrifugation at 12,000 rpm. For purification of the DNcSHMT protein, the clarified supernatant was applied directly to a CM Sepharose ion exchange column (Clontech), and the protein was purified to homogeneity as described previously (15). Recombinant SHMT from either bacteria that expressed the cSHMT protein or that coexpressed cSHMT and DNcSHMT proteins was purified by affinity chromatography. The cell suspensions were centrifuged at 12,000 rpm for 20 min at 4 °C to pellet-insoluble material. The cSHMT protein, which contains an N-terminal polyhistidine tag, was purified from the clarified sample on a Talon® metal affinity resin following the manufacturer's instructions (Clontech). The purity of all proteins was determined by SDS-polyacrylamide gel electrophoresis, and the protein concentrations were determined by a modified Lowry assay (16). The purified protein was stored at -80 °C.

SHMT Activity Assay-- Michaelis-Menten constants were determined for the cSHMT and DNcSHMT-catalyzed cleavage of allothreonine using the coupled enzyme assay with alcohol dehydrogenase as described previously (17). The rate of absorbance loss at 340 nm was recorded after the addition of 300-2000 pmol of SHMT to a 1-ml cuvette containing 25 mM HEPES, pH 7.2, 10 mM 2-mercaptoethanol, allothreonine, alcohol dehydrogenase, and 0.15 mM NADH in a spectrophotometer (Shimadzu UV-2401PC).

Affinity of the Recombinant DNcSHMT Protein for 5-FormylTHF-- The affinity of recombinant DNcSHMT protein for 5-formylTHF was determined by a previously described competitive binding assay (18). The binding of reduced folates to cSHMT results in the formation of a PLP-glycine-quinonoid intermediate, which has an absorption maximum at 502 nm (epsilon  = 40,000) (19). The DNcSHMT does not form the quinonoid intermediate and therefore did not exhibit an increase in absorbance at 502 nm upon binding 5-formylTHF. For the competitive binding assay, recombinant human cSHMT protein (10 µM) was added to a cuvette that contained 1 ml of the reaction buffer (200 mM glycine, 50 mM HEPES, pH 7.3, and 10 µM (6S) 5-formylTHF (a value equal to the Kd)). The absorbance spectrum was recorded from 550 to 400 nm. Recombinant DNcSHMT was added (to a final concentration of 25 µM), and the spectrum was recorded. To quantify the affinity of DNcSHMT for 5-formylTHF, the loss of absorbance at 502 nm was recorded as a function of DNcSHMT added to the cuvette as described previously (18).

Monomer Exchange Studies-- The ability of SHMT monomeric subunits to exchange among preformed cSHMT tetramers was determined by incubating purified, recombinant cSHMT protein (5 µM) in a solution containing 200 mM glycine, 50 mM HEPES, pH 7.3, and 200 µM (6RS) 5-formylTHF. The absorbance spectrum of the protein was recorded from 550 to 450 nm. Then DNcSHMT was added to a final concentration of 20 µM, and the spectra were recorded following 10-min, 1-h, and 24-h incubations at 37 °C. Loss of absorbance at 502 nm indicates that recombinant cSHMT monomers are exchanging with DNcSHMT monomers. To determine the effect of glycine or 5-formylTHF on monomer exchange, glycine and 5-formylTHF were omitted from the incubation solution. Following incubation, glycine (200 mM) and 5-formylTHF (200 µM) were added to the protein solution, and the absorbance spectrum was recorded immediately. For all of the experiments, the absorbance at 502 nm was recorded for the protein that underwent the exchange reaction and compared with the absorbance at 502 nm for purified cSHMT protein that did not undergo the exchange reaction.

Subunit Interchange with ApocSHMT Enzyme-- PLP was removed from the cSHMT active site by the addition of L-cysteine, which reacts with the bound PLP to form a thiazolidine complex (20). L-Cysteine (16 mg/ml) was added to a 2-ml solution that contained 3 mg of cSHMT (with an N-terminal polyhistidine tag), 20 mg of DNcSHMT, and 100 mM 2-mercaptoethanol. The solution was incubated at room temperature for 15 min. The protein was precipitated by the addition of ammonium sulfate to 70% saturation, incubated on ice for 5 min, and then centrifuged at 4300 rpm for 20 min. The precipitated protein pellet was suspended in 2 ml of 100 mM L-cysteine, 100 mM 2-mercaptoethanol. This cycle was repeated three times, and the protein was incubated at 37 °C for 5 min. The procedure lasted 3 h in duration. The protein was then dialyzed for 24 h against 2 liters of 20 mM potassium phosphate, pH 7.2, 2.5 mM 2-mercaptoethanol, 100 mM glycine, and 100 nM PLP at 4 °C. A control reaction contained 3 mg of cSHMT (with an N-terminal polyhistidine tag), 20 mg of DNcSHMT, and 100 mM 2-mercaptoethanol and was incubated at room temperature for the duration of the procedure described and stored at 4 °C for the duration of the dialysis described above. After 24 h, both the experimental and control proteins were dialyzed against a buffer containing 10 mM Tris-Cl, pH 7.0, 50 mM NaCl, 50 mM glycine, and 100 nM PLP for 1 h at 4 °C. The proteins were each purified using the batch/gravity flow protocol for the Talon® metal affinity resin (Clontech) with the following buffers: extraction/wash buffer (10 mM Tris-Cl, pH 7.0, 50 mM NaCl, 50 mM glycine, and PLP), stringent wash buffer (20 mM Tris-Cl, pH 7.0, 50 mM NaCl, 50 mM glycine, PLP, and 12.5 mM imidazole), and elution buffer (20 mM Tris-Cl, pH 7.0, 100 mM NaCl, 50 mM glycine, PLP, and 200 mM imidazole). Each protein was dialyzed overnight at 4 °C in 20 mM potassium phosphate, pH 7.2, 100 mM glycine, 3 mM 2-mercaptoethanol, and 100 nM PLP and then analyzed for exchange by SDS-PAGE. The protein bands were quantified using ChemiImager 4400 from Alpha Innotech Corp. (San Leandro, CA). This densitometry method was validated by analyzing a series of gels that contained 0.5-10 µg of cSHMT protein/lane. The optical density values increased linearly as a function of cSHMT concentrations from 0.5 to 4 µg cSHMT/lane.

SDS-Polyacrylamide Gel Electrophoresis-- Purified proteins (1-3 µg) were suspended in buffer containing 2% SDS, 62.5 mM Tris, pH 6.8, 100 mM dithiothreitol, and 10% glycerol and then incubated at 100 °C for 10 min. The purified proteins were then run on a mini-SDS-PAGE using a 5% stacking gel and 12% separating gel in a slab gel apparatus (Bio-Rad) with the discontinuous buffer system of Laemmli.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design of a Dominant-negative SHMT Protein-- To study the assembly of cSHMT tetramers and the occurrence of dynamic interchange of cSHMT subunits among cSHMT tetramers, a human DNcSHMT protein was designed using information derived from previous studies of mutated cSHMT proteins, as well as information derived from the murine cSHMT protein crystal structure (9). The murine cSHMT structure was solved with glycine and 5-formylTHF bound at the active site. The cSHMT-Gly-5-formylTHF ternary complex is an intermediate state analog of the cSHMT-Ser-THF catalytic complex (9), and this structure was used to design rationally a dominant-negative SHMT protein that can deactivate cSHMT activity.

The DNcSHMT protein was designed to: 1) oligomerize with and deactivate recombinant cSHMT monomeric subunits by inhibiting serine and allothreonine cleavage activity, 2) have decreased affinity for folate, and 3) retain affinity for PLP. Modeling studies indicated that three amino acid substitutions on a single cSHMT polypeptide were needed to achieve these goals, and these three amino acids are conserved in all known SHMT enzymes (Fig. 1A). Lys257 is the active site lysine in the murine and human cSHMTs that forms a Schiff base with the PLP cofactor. Mutation of this active site Lys to Gln inactivates the Escherichia coli cSHMT. The mutated protein can catalyze only a single turnover; this mutation does not allow the expulsion of the amino acid product, and therefore subsequent turnover is inhibited. However, this mutant retains affinity for folate cofactors and purifies with a PLP and a bound amino acid (20) (Table I). Tyr83 in the human and mouse cSHMT forms a hydrogen bond with the carboxylate of the amino acid substrate, and mutation of the analogous residue in the E. coli SHMT to Phe decreases the specific activity of the protein by greater than 99% and increases the affinity of the enzyme for tetrahydrofolate (Table I) (21). Tyr83 and Lys257 from the same polypeptide function in different active sites within the obligate dimer, and therefore dimerization between cSHMT and SHMT monomers that contain the double mutation, Y83F/K257Q, would be expected to lack catalytic activity in both subunits but retain folate binding in both subunits (Fig. 1). A third mutation was designed to eliminate folate binding to the DNcSHMT protein. The crystal structure of the murine SHMT protein shows that Tyr82 forms a stacking interaction with the p-aminobenzoylglutamate moiety of THF that is predicted to be essential for THF binding (Fig. 1), although this has not been tested experimentally (9).


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Fig. 1.   Schematic representation of the cSHMT active sites that result from association of cSHMT and DNcSHMT monomers. Amino acid residues that contribute to the active site and were the targets for mutagenesis are shown in boxes. Residues that contribute to an active site that originate from the opposite monomer within the obligate dimer are indicated with a prime symbol. A, active site of a cSHMT homodimer with PLP, glycine, and bound 5-formylTHF. B, active site of a DNcSHMT homodimer with three mutations in the active site. C and D, the two active sites that result from heterodimer formation between a cSHMT and DNcSHMT monomer.


                              
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Table I
Predicted properties of the DNcSHMT based on studies of the E. coli SHMT

The DNcSHMT monomer was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F (Fig. 1B). Purified recombinant DNcSHMT is expected to retain high affinity for PLP in the presence of amino acid substrates but to lack serine cleavage catalytic activity and affinity for folate cofactors. Oligomerization of the DNcSHMT monomer with a cSHMT monomer will generate two active sites (Fig. 1, C and D). Active site C lacks Lys257 and therefore should lack catalytic activity but retain affinity for folate cofactors. Site D lacks Tyr82 and Tyr83 and therefore is anticipated to have less than 1% serine cleavage activity and decreased affinity for folate cofactors.

Expression and Characterization of the DNcSHMT Recombinant Protein-- The DNcSHMT and cSHMT proteins were coexpressed in E. coli to test the ability of the DNcSHMT monomers to oligomerize with and inactive cSHMT protein as illustrated in Fig. 1. The human recombinant cSHMT cDNA was engineered with an N-terminal polyhistidine tag to enable affinity purification of this protein; the DNcSHMT cDNA lacked a coding sequence for the polyhistidine tag. Fig. 2A (lanes 1 and 2) shows that both the cSHMT and DNcSHMT proteins can be expressed in E. coli and that the cSHMT protein can be separated from the DNcSHMT protein by SDS-PAGE because of its increased molecular mass resulting from the polyhistidine tag. Fig. 2A (lane 3) shows that the cSHMT protein is more abundant in crude extracts of E. coli that coexpress the cSHMT and DNcSHMT proteins. Following purification, all of the recombinant proteins were greater than 95% pure (Fig. 2B, lanes 1-3). As expected, affinity purification of cSHMT protein from E. coli that coexpressed the cSHMT and DNcSHMT proteins also resulted in the purification of DNcSHMT protein, indicating that the cSHMT and DNcSHMT subunits associate with one another. Additionally, the ratio of cSHMT to DNcSHMT monomeric subunits in purified cSHMT/DNcSHMT protein was 1.0, despite the higher concentration of cSHMT protein compared with DNcSHMT protein in the crude extracts. This indicates that cSHMT monomers may have higher affinity for DNcSHMT monomers than other cSHMT monomers.


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Fig. 2.   SDS-PAGE gels of recombinant human cSHMT and DNcSHMT proteins. A is a crude protein extract from E. coli expressing the cSHMT protein with an N-terminal polyhistidine tag (lane 1), the DNcSHMT protein without a polyhistidine tag (lane 2), or both the cSHMT and DNcSHMT proteins (lane 3). B shows the purified SHMT proteins. Lane 1, cSHMT protein with a polyhistidine tag (purified on Talon® affinity column); lane 2, DNcSHMT protein that lacks a polyhistidine tag (purified on CM Sephadex); lane 3, coexpressed DNcSHMT and cSHMT proteins (purified on a Talon® affinity column). Approximately 0.85 µg of purified protein was run on a 12% mini-SDS-PAGE using the discontinuous buffer system of Laemmli. The gel was stained with SimplyBlue (Invitrogen) to visualize the proteins.

Prediction of Subunit Assembly-- Two models were derived to predict the assembly and catalytic activity of cSHMT/DNcSHMT heterotetramers (Fig. 3). Model I is a random association model whereby cSHMT and DNcSHMT monomers randomly associate to form heterodimers and homodimers, which randomly associate to form homotetramers and heterotetramers (Fig. 3, model I). Model 2 predicts that cSHMT and DNcSHMT monomers can only form homodimers but that homodimers can randomly associate to form homotetramers and heterotetramers. Assuming equal concentrations of cSHMT and DNcSHMT monomers (as shown in Fig. 2), the expected frequency of each potential tetramer was calculated for models I and II. Additionally, the activity of each tetramer was predicted by assuming that both DNcSHMT homodimers and DNcSHMT/cSHMT heterodimers are inactive and that the formation of tetramers from obligate dimers does not influence the activity of either obligate dimer. The expected specific activity of the purified cSHMT/DNcSHMT protein will be the average of the specific activity for each tetrameric isoform, after correcting for its relative abundance. If the random association model is correct (model I), we anticipate that the specific activity of the cSHMT/DNcSHMT tetrameric protein will be reduced by 75% compared with tetrameric cSHMT protein. For model II, the cSHMT/DNcSHMT tetrameric protein is predicted to exhibit a 50% decrease in specific activity compared with cSHMT protein.


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Fig. 3.   Models that predict the assembly and activity of cSHMT/DNcSHMT obligate dimers and tetramers. The cSHMT monomers are shown as open circles, and the DNcSHMT monomers are shown as gray circles. WT denotes a nonmutated cSHMT monomer, and DN denotes a DNcSHMT monomer. Activity predictions assume that cSHMT/DNcSHMT dimers are inactive and that obligate dimers do not communicate or influence each other within the tetramer.

Spectral Properties of cSHMT-- The cSHMT protein is a PLP-dependent enzyme, and the reaction intermediates associated with catalysis have distinct spectral properties. PLP binds to cSHMT through a Schiff base with Lys257 forming an intermediate known as the internal aldimine (Fig. 4, structure 1). Binding of amino acid substrates results in the formation of the geminal diamine (Fig. 4, structure 2), which is a tetrahedral intermediate that results from partial displacement of Lys257 by the incoming amino acid. Full displacement of Lys257 results in a Schiff base between the amino acid substrate and the active site PLP, an intermediate known as the external aldimine (Fig. 4, structure 3). Loss of the pro-2S proton of glycine, or the hydroxymethyl group of serine, results in the formation of a highly conjugated glycine quinonoid intermediate (Fig. 4, structure 4).


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Fig. 4.   Structures of catalytic intermediates associated with the SHMT serine retroaldol cleavage mechanism.

Fig. 5A shows the spectrum of the cSHMT-Gly binary complex (spectrum 1) and the cSHMT-Gly-5-formylTHF ternary complex (spectrum 2). The absorbance spectrum of the cSHMT-glycine binary complex shows the presence of the external aldimine (lambda max = 425 nm) and the glycine quinonoid (lambda max = 492 nm). The addition of 5-formylTHF shifts the equilibrium of the enzyme-bound PLP to the glycine quinonoid (lambda max = 502 nm). Fig. 5B shows the spectrum of the DNcSHMT-Gly binary complex (spectrum 1) and that of the DNcSHMT-Gly binary complex in the presence of 5-formylTHF (spectrum 2). Previous studies have demonstrated that the Lys to Gln mutation does not permit formation of the internal aldimine or geminal diamine (20), and the concentration of the glycine quinonoid associated with the cSHMT-Gly-5-formylTHF ternary complex is reduced to less than 0.1% compared with the cSHMT protein (Table I). Other studies have shown that the Tyr to Phe mutation eliminates the formation of the glycine quinonoid (Table I) (21). The spectra of the DNcSHMT-Gly binary complex in the presence and absence 5-formylTHF are consistent with previous studies because no quinonoid intermediate was seen. Fig. 5C shows the spectra of the cSHMT/DNcSHMT-Gly binary complex (spectrum 1) and the cSHMT/DNcSHMT-Gly-5-formylTHF ternary complex (spectrum 2). The concentration of the quinonoid intermediate associated with the cSHMT/DNcSHMT-Gly-5-formylTHF ternary complex was decreased by 75% compared with the concentration of the quinonoid intermediate in the cSHMT-Gly-5-formylTHF ternary complex. The formation of a quinonoid intermediate is a measure of catalytic competence, and the 75% reduction in the concentration of the quinonoid intermediate in the cSHMT/DNcSHMT-Gly-5-formylTHF complex is consistent with random association of cSHMT and DNcSHMT monomeric subunits within the tetramer (Fig. 2, model I). This result indicates that the K257Q, Y82A, and Y83F mutations inactivate the cSHMT enzyme and that DNcSHMT deactivates the cSHMT protein as predicted.


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Fig. 5.   Spectral properties of the cSHMT enzymes. The absorbance spectra of the SHMT enzymes (25 µM) was determined in the presence of 200 mM glycine (spectrum 1) and 200 mM glycine, 200 µM (6RS)5-formylTHF (spectrum 2). A, recombinant human cSHMT homotetramers; B, recombinant human DNcSHMT homotetramers; C, recombinant human cSHMT/DNcSHMT heterotetramers.

Affect of Y82A on Folate Binding-- The Y82A mutation in the DNcSHMT protein is predicted to reduce the affinity of cSHMT for 5-formylTHF. The ability of DNcSHMT to bind folate was investigated using a competitive binding assay described elsewhere (Fig. 6) (18). In this assay, a solution containing cSHMT protein (10 µM), saturating concentrations of glycine (200 mM), and 5-formylTHF (10 µM, the concentration equal to the Kd) is titrated with the DNcSHMT. Loss of absorbance at 502 nm following the addition of DNcSHMT would indicate that DNcSHMT binds 5-formylTHF. The intensity of the glycine quinonoid intermediate was not diminished by the addition of up to 25 µM DNcSHMT to this solution, indicating that DNcSHMT does not have a high affinity for folate cofactors (Fig. 6).


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Fig. 6.   Affinity of the DNcSHMT homotetramers for 5-formylTHF. Purified recombinant cSHMT protein (10 µM) was incubated in a solution of 20 mM potassium phosphate, pH 7.2, 200 mM glycine, and 10 µM (6S)5-formylTHF (equal to the value of Kd), and spectrum 1 was recorded. The DNcSHMT protein was added to a final concentration of 20 µM, and spectrum 2 was recorded after a 60-min incubation at 28 °C. No decrease in the absorbance at 502 nm was observed following the addition of DNcSHMT protein, indicating that the DNcSHMT protein does not bind folate. The increase in the absorbance at 502 nm following the 60-min incubations occurred independently of the addition of DNcSHMT protein.

A similar experiment was performed to determine whether cSHMT monomers exchange among preformed cSHMT and DNcSHMT tetramers. The competitive binding experiment described above was repeated with two alterations: (6RS)5-formylTHF was added in saturating concentrations (200 µM), and the incubation time for the reaction was extended to 24 h at 37 °C. After 24 h, no decrease in the absorbance at 502 nm occurred, indicating that cSHMT and DNcSHMT monomers do not exchange from preformed cSHMT and DNcSHMT tetramers. To determine whether monomer exchange occurred from preformed cSHMT and DNcSHMT tetramers in the absence of glycine and 5-formylTHF, a solution containing cSHMT (10 µM) and DNcSHMT (20 µM) tetramers was incubated at 37 °C for 24 h, then glycine and 5-formylTHF were added to the reaction, and the absorbance intensity at 502 nm was determined. The absorbance at 502 nm was identical to that observed for a solution of cSHMT (10 µM) incubated without DNcSHMT, indicating that cSHMT and DNcSHMT monomers do not exchange from preformed tetramers in the presence or absence of glycine and 5-formylTHF.

Effect of PLP on Subunit Interchange among cSHMT and DNcSHMT Tetramers-- In this study, the affect of PLP on the exchange of cSHMT subunits between preformed cSHMT and DNcSHMT tetramers was investigated as described under "Experimental Procedures." Incubation of purified, recombinant cSHMT protein with a 7-fold molar excess DNcSHMT followed by affinity purification of the cSHMT protein did not result in the copurification of DNcSHMT, indicating that cSHMT subunits do not exchange between preformed cSHMT and DNcSHMT tetramers when PLP is bound (Fig. 7, lane 1). However, when cSHMT and DNcSHMT proteins that lack bound PLP are incubated together, subunit exchange must have occurred because DNcSHMT copurified with cSHMT on the affinity column (Fig. 7, lane 2). The ratio of cSHMT to DNcSHMT monomers in the purified protein was 63% to 27%, indicating that the subunit exchange had not reached equilibrium. Incubation beyond 3 h was not possible because of the formation of insoluble precipitate, suggesting that cSHMT dimers are unstable.


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Fig. 7.   Effect of PLP on SHMT subunit exchange. The cSHMT (3 mg) and DNcSHMT (20 mg) proteins were coincubated for 3 h, then the cSHMT protein was purified on a Talon® affinity column, and the presence of DNcSHMT was determined by SDS-PAGE as described under "Experimental Procedures." Lane 1, purified cSHMT following incubation of cSHMT and DNcSHMT protein; lane 2, purified cSHMT protein following incubation of apocSHMT and apoDNcSHMT protein that lacks PLP; lane 3, Purified cSHMT protein from E. coli that coexpressed DNcSHMT and cSHMT proteins. Approximately 1-3 µg of purified protein was run on a 12% mini-SDS-PAGE using the discontinuous buffer system of Laemmli. The gel was stained with SimplyBlue (Invitrogen) to visualize the proteins.

Because cSHMT monomers have never been isolated and loss of PLP results in the dissociation of tetramers to dimers, it is assumed that loss of PLP permits exchange of obligate homodimers between cSHMT and DNcSHMT tetramers (Fig. 3, model II). To test this hypothesis, absorbance spectra of the purified protein that underwent the subunit exchange reaction were recorded (Table II). The addition of glycine and 5-formylTHF to this purified protein resulted in the formation of the glycine quinonoid intermediate, with an A502 nm that was 30% less than that observed for the cSHMT homotetramer (Table II), indicating that the predicted catalytic competency of the cSHMT enzyme had decreased 30% following the exchange reaction. This 30% reduction in cSHMT predicted catalytic competency represents the percentage of DNcSHMT subunits in the tetramer after the exchange reaction (Fig. 7), indicating that these cSHMT/DNcSHMT heterotetramers are comprised of cSHMT and DNcSHMT homodimers (Fig. 3, model II). It should be noted that the dimer exchange reaction did not go to completion and that a 50% decrease in predicted catalytic competency would be the maximum expected if the reaction went to completion. Monomer exchange could not have occurred because no dominant-negative effect was seen, thus the results are only consistent with model II. This study demonstrates that the activity of cSHMT obligate dimers is not affected by tetramerization with DNcSHMT obligate dimers, a primary assumption in our models (Fig. 3).


                              
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Table II
Effect of DNcSHMT on cSHMT catalytic activity

Catalytic Activity of cSHMT and DNcSHMT Proteins-- The catalytic activities of the cSHMT, DNcSHMT, and DNcSHMT/cSHMT tetramers were determined to verify that quinonoid formation was an adequate indicator of cSHMT catalytic potential. The results shown in Table II verify that the catalytic activity (kcat) of the enzymes parallel their ability to form glycine quinonoid intermediates and that the DNcSHMT protein can only deactivate cSHMT catalytic function when it dimerizes with cSHMT monomers.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The generation of a DNcSHMT protein enabled a thorough investigation into the dynamic exchange of subunits among cSHMT tetramers and the ability of amino acids, folates, and PLP to affect subunit exchange among preformed cSHMT tetramers. Furthermore, the study of cSHMT/DNcSHMT heterotetramers gives new insight into the stoichiometry of folate binding and communication among active sites within the tetramer.

Pyridoxal Phosphate Inhibits Dynamic Subunit Exchange-- A polymorphism in the cSHMT gene is associated with altered serum homocysteine levels (a risk for birth defects and certain cancers) (22) and decreased risk for leukemia (23). Therefore, understanding the factors that regulate cSHMT activity is important to elucidate the mechanisms whereby altered folate metabolism influences risk for diseases and birth defects (1). SHMT activity is decreased in the livers of vitamin B6-deficient rats, and it is presumed that the decreased activity results from the loss of PLP cofactor from the active site and formation of apoenzyme (24). However, the effect of vitamin B6 deficiency on SHMT turnover rates has not been investigated. Whereas other studies have demonstrated that loss of PLP from the SHMT active site weakens the interactions of the obligate dimers within the tetramer, this study demonstrates that cSHMT tetramers are very stable and do not exchange subunits unless they lack bound PLP because PLP inhibits this exchange by stabilizing the tetramer. Further investigation is required to determine whether cellular PLP deficiency results in increased rates of cSHMT protein turnover resulting from dissociation of cSHMT tetramers.

Communication among cSHMT Monomers and Obligate Dimers-- Previous titration calorimetry studies have demonstrated that only half of the active sites within the cSHMT tetramer bind reduced folates (25). There are three mechanisms that may account for half-site occupancy within the cSHMT tetramer: 1) half-site occupancy within the obligate dimer, 2) asymmetric obligate dimers with one obligate dimer saturated with folate, the other dimer lacking folate, or 3) random binding of two folate molecules/SHMT tetramer. Mechanism 1 implies that only active sites within the obligate dimer communicate, whereas mechanisms 2 and 3 assume that all four active sites within a tetramer communicate. Mechanism 1 is supported by the report of the Bacillus stearothermophilus SHMT crystal structure (12). This enzyme is an obligate dimer in solution because it lacks the amino acid residues required for tetramer formation. This enzyme was crystallized with glycine and 5-formylTHF bound, and only one of the active sites within the obligate dimer contained 5-formylTHF. In contrast, the structure of the E. coli cSHMT that was solved with 5-formylTHF and bound glycine does not support mechanism 1 (11); this structure showed 5-formylTHF tightly bound in both active sites of the obligate dimer (11). Mechanism 2 is supported by the report of the E. coli (described above) and murine cSHMT structures that were obtained from crystals grown in the presence of glycine and 5-formylTHF (9). The mouse structure had only two equivalents of 5-formylTHF bound tightly, with one obligate dimer displaying full occupancy of the active sites, and no folate binding or disordered folate binding in the other obligate dimer (9), indicating negative cooperativity between the obligate dimers within the tetrameric enzyme. There are no data to support mechanism 3.

The inability of obligate dimers to communicate with each other (mechanism 1) within the cSHMT tetramers was an underlying assumption of our assembly models (Fig. 3), and these assumptions were supported by the experimental data presented. The data presented here also support mechanism 1 with respect to the stoichiometry of folate binding within the tetramer. Loss of PLP results in the exchange of obligate dimers between preformed cSHMT and DNcSHMT tetramers (Fig. 3, model 2) and a decrease in the specific activity of the cSHMT protein (Table II). The 34% decrease in the specific activity associated with cSHMT/DNcSHMT heterotetramer formation is fully accounted for by the inclusion of inactive DNcSHMT obligate dimers within the tetramers (Fig. 7), thereby diluting the specific activity of the cSHMT homodimers that exchanged with DNcSHMT homodimers by 50%. If mechanism 2 for folate binding is correct, the concentration of the quinonoid should be the same in tetramers that are formed from two cSHMT homodimers and from cSHMT/DNcSHMT tetramers formed from cSHMT homodimers and DNcSHMT homodimers (Fig. 3, model 2). The replacement of an "inactive cSHMT obligate dimer" (that cannot bind folate) within the cSHMT homotetramers with an inactive DNcSHMT obligate dimer should not alter the intensity of the quinonoid within the tetramer. Furthermore, "inactive cSHMT obligate dimers" should become "activated" following tetramer formation with DNcSHMT homodimers. Therefore, these studies seem to eliminate mechanism 2 with respect to folate binding to cSHMT tetramers in solution. We recognize that this mechanism is not consistent with results from the E. coli and murine SHMT structures, but these results are supported by the structure of the B. stearothermophilus cSHMT enzyme (12). Also, we recognize that the ability of cSHMT obligate dimers to communicate within the tetramer may have been lost as a result of minor structural perturbations resulting from the three mutations in the DNcSHMT protein.

DNcSHMT Inactivates cSHMT Activity-- The data presented here demonstrate that the DNcSHMT monomers can effectively and randomly associate with cSHMT monomers and deactivate them, indicating that this construct may be effective in inhibiting cSHMT function and one-carbon metabolism in vivo. The data also indicate that the DNcSHMT protein can only inhibit cSHMT activity by forming heterodimers, and therefore the DNcSHMT cannot inhibit the activity of preformed cSHMT tetramers. Previously, we have demonstrated that cSHMT plays a key role in one-carbon metabolism by accelerating de novo thymidylate biosynthesis and also by inhibiting homocysteine remethylation in MCF-7 cells (1). We are currently generating cancer cell lines and transgenic mice that express the DNcSHMT protein to further understand the metabolic role of cSHMT. This approach could also be used to deactivate the mitochondrial SHMT isozyme because these three mutated residues within the DNcSHMT protein are conserved in all SHMT proteins.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant DK58144 (to P. J. S.) and Training Grant DK07158-21 (to K. A. Z.).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.

Dagger To whom correspondence should be addressed: Cornell University, 315 Savage Hall, Ithaca, NY 14853. Tel.: 607-255-9751; Fax: 607-255-9751; E-mail: PJS13@cornell.edu.

Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211569200

    ABBREVIATIONS

The abbreviations used are: SHMT, serine hydroxymethyltransferase; 5-formylTHF, 5-formyltetrahydrofolate; PLP, pyridoxal 5'-phosphate; cSHMT, cytoplasmic serine hydroxymethyltransferase; DNcSHMT, dominant-negative cytoplasmic serine hydroxymethyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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