 |
INTRODUCTION |
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 |
Materials--
5-FormylTHF, allothreonine, alcohol
dehydrogenase, NADH, isopropyl
-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
-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 (
= 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 |
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).

View larger version (28K):
[in this window]
[in a new window]
|
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.
|
|
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.

View larger version (55K):
[in this window]
[in a new window]
|
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.

View larger version (30K):
[in this window]
[in a new window]
|
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).

View larger version (15K):
[in this window]
[in a new window]
|
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 (
max = 425 nm) and the
glycine quinonoid (
max = 492 nm). The addition of
5-formylTHF shifts the equilibrium of the enzyme-bound PLP to the
glycine quinonoid (
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.

View larger version (12K):
[in this window]
[in a new window]
|
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).

View larger version (11K):
[in this window]
[in a new window]
|
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.

View larger version (10K):
[in this window]
[in a new window]
|
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).
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 |
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.