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(Received for publication, October 27, 1994) From the
The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH)
(EC 1.5.1.6) catalyzes both the NADP 10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) ( All known
aldehyde dehydrogenases contain a conserved cysteine, which, in the
case of 10-FTHFDH, is Cys-707(4, 5) . The conserved
cysteine is presumed to act as a nucleophile in the formation of an
enzyme-linked thiohemiacetal intermediate(6, 7) . A
number of different approaches may be used to identify the amino acid
at the active site of an enzyme. Using an affinity label, Blatter et al.(7) found that Cys-302 formed a covalent
intermediate with the substrate in human aldehyde
dehydrogenase(7) . Site-directed mutagenesis confirmed that
Cys-302 is essential for enzyme activity of rat liver mitochondrial
aldehyde dehydrogenase, whereas Cys-49 and Cys-162 can be changed to
alanine without altering enzyme activity(6) . Additionally,
site-directed mutagenesis experiments have shown that serine 74 is also
important for enzyme activity(6) . This observation was
unexpected because this serine is conserved only in mammalian aldehyde
dehydrogenases(5) . It has been shown recently that serine 74
is not involved in aldehyde oxidation but may be involved in
NAD
Figure 1:
Construction of the vector for
expression of rat 10-FTHFDH. A, subcloning 10-FTHFDH cDNA from
pBS KS
Aldehyde dehydrogenase activity was assayed
using propanal essentially as described by Lindahl and
Evces(23) . The reaction mixture contained 60 mM sodium pyrophosphate buffer, pH 8.5, 5 mM propanal, 1
mM NADP
Figure 2:
Activity of wild type and mutant
10-FTHFDH. HYD, hydrolase activity; DH, dehydrogenase
activity; TOTAL, total activity (measures both dehydrogenase
and hydrolase activity). The assays were performed as described under
``Experimental Procedures.'' A,
10-HCO-H
It has been shown that 10-FTHFDH is able to
oxidize a series of aldehyde substrates in the presence of
NADP(4) . Kinetic analysis of propanal oxidation by recombinant
10-FTHFDH showed a K Analysis of
activity of mutant 10-FTHFDH in the absence of NADP
Figure 3:
Fluorescence titration of 10-FTHFDH with
NADP
The sequence identity between 10-FTHFDH and the group of
aldehyde dehydrogenases suggested that Cys-707 could be the active site
nucleophile. In order to test this hypothesis, Cys-707 in 10-FTHFDH was
modified. The closest amino acid substitution for cysteine is serine.
Serine can, however, function as a nucleophile in place of cysteine in
some enzyme-catalyzed reactions(27) . Therefore we replaced
Cys-707 with alanine. Such a substitution is used often in
site-directed mutagenesis to study the role of cysteine residues. In
our experiments mutation of Cys-707 of 10-FTHFDH to Ala resulted in
complete loss of dehydrogenase activity of the enzyme for both the
native substrate 10-HCO-H Substitution of
a single amino acid residue can sometimes result in a decrease of
enzyme activity even if this residue is not involved in the active site
due to changes in the higher order protein structure(28) . Such
critical residues play a basic role in protein folding and/or in
supporting correct protein structure(29, 30) .
Replacement of such residues could lead to decreased protein stability (31) and also to a decreased level of expression due to higher
accessibility of mutant proteins to proteases(32) . On the
other hand, many amino acid substitutions do not have large effects on
protein stability(33, 34) . Even when a replaced
residue is essential for protein conformation, protein structures
adjust to compensate for changes in sequence(29) . Our study
did not reveal any changes in stability of the C707A mutant in
comparison with the wild type protein and both displayed a similar
level of expression. Moreover, the K The mutation of Cys-707 to Ala did not produce any
changes in the hydrolase activity of 10-FTHFDH. This indicates that the
enzyme has two different catalytic sites. This observation is not
surprising since Rios-Orlandi et al.(2) showed that
both dehydrogenase and hydrolase activities can take place
simultaneously. The existence of two different functional domains was
predicted when the sequence of the enzyme was determined(4) . A
recent study by Schirch et al.(35) where
differential scanning calorimetry and proteolytic digestion were used
has also shown the hydrolase and dehydrogenase activities of rabbit
liver 10-FTHFDH reside in different domains. The results show that
cysteine 707 is a key residue of the dehydrogenase active site of
10-FTHFDH. Similar results were obtained for aldehyde dehydrogenase
from rat liver mitochondria(6) , which has 46% homology with
the carboxyl-terminal domain of rat 10-FTHFDH(4) . Aldehyde
dehydrogenase with the corresponding C302A mutation was devoid of
activity(6) . At the same time substitution an alanine for 2
other cysteines in positions 49 and 162 of aldehyde dehydrogenase did
not result in changes of enzyme activity(6) . Apparently, the
dehydrogenase catalytic site of 10-FTHFDH is structurally similar to
that of aldehyde dehydrogenase with a cysteine as a catalytic
nucleophile residue and supports the role of a thiohemiacetal
intermediate in the reaction mechanism.
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 519-522
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-dependent
oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO
and the NADP-independent hydrolysis of
10-formyltetrahydrofolate to tetrahydrofolate and formate. The
COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a
46% identity with a series of NAD
-dependent aldehyde
dehydrogenases (EC 1.2.1.3). All known members of the aldehyde
dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine
(Cys-707 for 10-FTHFDH), which has been predicted to be at the active
site of these enzymes. Rat liver 10-FTHFDH was expressed in a
baculovirus system, and site-directed mutagenesis has been used to
study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH
with alanine substituted for cysteine at position 707 had no
dehydrogenase activity, while hydrolase activity and binding of
NADP
were unchanged. Light scattering analysis
revealed that wild type and mutant 10-FTHFDH exist as tetramers. We
conclude that cysteine 707 is directly involved in the active site of
10-FTHFDH responsible for dehydrogenase activity, and there is a
separate site for the hydrolase activity.
)(EC 1.5.1.6) catalyzes both the
NADP-dependent oxidation of 10-formyltetrahydrofolate
(10-HCO-H
PteGlu) to tetrahydrofolate and CO and
the NADP-independent hydrolysis of
10-HCO-H
PteGlu to tetrahydrofolate and
formate(1, 2, 3) . The amino-terminal domain
of rat liver 10-FTHFDH (residues 1-203) is 24-30% identical
to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.1)
from different species(4) . The carboxyl-terminal domain
(residues 417-902) of 10-FTHFDH has 46% identity with a series of
NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3),
and the enzyme is able to oxidize aldehydes(4) .
binding(8) . In the present study we
expressed rat 10-FTHFDH in a baculovirus system and used site-directed
mutagenesis to elucidate the role of the highly conserved Cys-707 in
the activity of the enzyme.
Materials
10-Formyl-5,8-dideazafolate (10-FDDF)
was obtained from Dr. John B. Hynes, Dept. of Pharmaceutical Chemistry,
Medical University of South Carolina.
(6R,S)-10-HCO-HPteGlu was prepared from
(6R,S)-5-HCO-HPteGlu (Sigma) by the
method of Rabinowitz(9) . Oligonucleotides were synthesized by
Research Genetics (Huntsville, AL). Restriction enzymes were purchased
from New England BioLabs, Inc. (Beverly, MA) or from Stratagene (La
Jolla, CA). Grace's insect cell medium and baculovirus agarose
were obtained from Invitrogen (San Diego, CA). Fetal bovine serum was
purchased from Intergen (Purchase, NY). Other chemicals were obtained
from Sigma.Vector Construction and Enzyme Expression
A PCR
fragment including 277 base pairs of the 5`-coding sequence of
10-FTHFDH from the start codon to a unique NcoI restriction
site and having an XbaI restriction site inserted at the
5`-end was synthesized using two oligonucleotides
5`-CCGGCCCATGGGTATGAACTGGCTGCAGAAGG-3` and
5`-CCGGCTCTAGAATGAAGATTGCAGTAATCGGA-3` by PCR using a Gene Amp kit
(Perkin Elmer, Norwalk, CT). The cDNA of rat liver 10-FTHFDH (4) was subcloned from pBS KS II into the pVL
1393 baculovirus vector (Invitrogen) through the unique EcoRI
restriction site. A fragment between the unique XbaI
restriction site of pVL 1393 and the unique NcoI restriction
site in the 10-FTHFDH cDNA sequence, including the whole 5`-noncoding
sequence of cDNA was removed and replaced with the XbaI-NcoI PCR-generated fragment, containing the
coding sequence (Fig. 1). The sequence of the construct was
confirmed by the dideoxynucleotide chain termination method (10) using a Sequenase DNA sequencing kit (U. S. Biochemical
Corp.). This construct was expressed in Sf9 cells, which were grown in
monolayer using the MaxBac expression system (Invitrogen) according to
the manufacturer's directions.
II (4) to pVL 1393 baculovirus vector; B, removal of the XbaI-NcoI 5`-end of the
cDNA including entire uncoding sequence; C, insertion of XbaI-NcoI PCR-generated fragment including coding
sequence only.
Mutant Construction
Mutation of the 10-FTHFDH was
achieved by oligonucleotide-directed mutagenesis. The codon for
cysteine, TGC, at position 707 was altered to GCC to produce an
alanine-containing mutant. The Transformer site-directed mutagenesis
kit (Clontech, Palo Alto, CA), based on the method of Deng and
Nickoloff(11) , was used to introduce the mutation into the pVL
1393-10-FTHFDH construct. Two oligonucleotides were used, the
mutagenic oligonucleotide 5`-CAAAGGGGAGAACGCCATTGCGGCAG-3` for
introduction of the mutation in 10-FTHFDH sequence and a selection
oligonucleotide 5`-GAATTCCGGAGAGGCCTCTGCAGATC-3` for conversion of one
unique restriction site, XmaIII to StuI, which was
also unique, in pVL 1393. The mutagenic experiments were carried out
according to the Clontech protocol. Introduction of the mutation was
confirmed by sequencing(10) . The mutant construct was
expressed in Sf9 cells similar to that for wild type protein.Analysis of Recombinant
10-FTHFDH
SDS-electrophoresis of 10-FTHFDH was done according to
the method of Laemmli(12) . Western immunoblot analysis was
performed as described by Burnette (13) with rabbit polyclonal
antiserum against pure rat liver 10-FTHFDH and goat anti-rabbit IgG
conjugated to alkaline phosphatase (Bio-Rad)(14) . The mutant
protein showed the same level of expression as wild type recombinant
10-FTHFDH. Analysis of culture medium and cell extract revealed that
most of the mutant enzyme was released from cells into the culture
medium, similar to the wild type recombinant enzyme, possibly due to
lysis of the cells, which occurs in the later post-infection
period(15) . More than 80% of the total enzyme was found in the
culture medium. Immunoblotting with specific anti-10-FTHFDH antiserum
showed only one major band corresponding to the enzyme itself,
indicating no proteolytic degradation of the enzyme during production.Purification of Recombinant 10-FTHFDH
The enzyme
was purified from the culture medium by affinity chromatography on a
column of Sepharose-5-formyltetrahydrofolate(16, 17) .
A column (1.5 
10 cm) was packed with about 8.0 ml of settled
gel and equilibrated with 0.01 M potassium phosphate buffer,
pH 7.0, containing 10 mM 2-ME and 1 mM NaN
(buffer 1). Medium (200 ml), plus 2-ME and NaN at 10
and 1 mM, respectively, was applied to the affinity column.
The column was then washed with buffer 1 (100 ml) followed by the same
buffer containing 1 M KCl (100 ml). The enzyme was then eluted
from the column with buffer 1 containing 1 M KCl and 20 mM folic acid. The eluate was passed through a column of Bio-Gel
P6-DG (Bio-Rad) equilibrated with buffer 1 at pH 6.2 to remove excess
folate. The eluate was concentrated to approximately 5 ml using an
Amicon (Beverly, MA) filtration cell. Additional purification was done
on a DE-52 column with the use of a linear NaCl gradient (0-0.5 M). The enzyme peak was eluted at 0.21 M NaCl,
collected, desalted on a column of Bio-Gel P6-DG, and stored at 4
°C. The mutant enzyme was purified by the same procedure. Affinity
chromatography resulted in purification of both wild type and mutant
recombinant protein from most of the proteins of bovine serum.
Additional purification on a DE-52 column gave homogeneous preparations
for both proteins.Molecular Size Determination
A molecular size
detector (model DynaPro-801, Protein Solutions, Inc., Charlottesville,
VA) employing laser-light scattering methodology (18, 19, 20) was used to analyze recombinant
10-FTHFDH under non-denaturing conditions. The enzyme (in 0.01 M potassium phosphate buffer, pH 7.0, containing 10 mM 2-ME
and 1 mM NaN) was concentrated using a Centricon
30 (Amicon) to about 1 mg/ml. Aliquots of this solution (200 µl)
were filtered (0.45 µm; Millipore Co., Bedford, MA) to remove any
dust particles prior to measurement. Each sample was measured for 20
cycles and the results analyzed using AutoPro 801 Data Analysis
Software (Protein Solutions).Measurement of Enzyme Activity
All assays were
performed at 30 °C in a Perkin-Elmer Lambda 4B double beam
spectrophotometer (Norwalk, CT). For measurement of hydrolase activity,
the reaction mixture contained 0.05 M Tris-HCl, pH 7.8, 100
mM 2-ME, and varying amounts of substrate, either
10-HCO-HPteGlu or 10-FDDF (0.5-18 µM).
The enzyme (1 µg) was added in a final volume of 1.0 ml. The
reaction was started by the addition of enzyme and read against a blank
cuvette containing all components except enzyme. Appearance of product
was measured at either 295 nm for 5,8-dideazafolate or 300 nm for
HPteGlu using molar extinction coefficients of 18.9
10 and 21.7 10 for 5,8-dideazafolate (21) and HPteGlu(22) , respectively.
Addition of NADP to the reaction mixture provided a
measure of both dehydrogenase and hydrolase activity, i.e. total activity of the enzyme. Hydrolase activity measured in the
absence of NADP
was subtracted from the total activity
to give the dehydrogenase activity. Dehydrogenase activity was also
measured independently using the increase in absorbance at 340 nm due
to production of NADPH and the molar extinction coefficient of 6.2
10., and enzyme in a total volume of 1
ml. Activity was estimated from the increase in absorbance at 340 nm.
Fluorescence Studies
Binding of NADP to 10-FTHFDH was detected by measuring the quenching of enzyme
fluorescence. These experiments were done on a Perkin-Elmer model
650-40 fluorescence spectrophotometer. Protein samples (9.0
nM) were in 50 mM Tris-HCl, pH 7.8, and 50 mM 2-ME. The NADP
concentration was varied from 0.02
µM to 10 µM. Fluorescence excitation was at
291 nm, and the emission was monitored at 340 nM(24) . K
for NADP was calculated from
data on fluorescence quenching in the presence of
NADP
, which were plotted in a linear
form(25) .
Analysis of 10-FTHFDH Oligomeric
Structure
Native 10-FTHFDH has been reported to exist as a dimer (16) or tetramer(2, 3) . Using a light
scattering technique, we investigated the oligomeric structure of wild
type and mutant recombinant 10-FTHFDH. The analysis showed that both
enzymes form monodisperse solutions with a Stokes radius of 7.63 nm and
estimated molecular mass of 398.77 kDa for the wild type enzyme and
7.53 nm and 393.15 kDa for the mutant enzyme. This indicates that both
wild type and mutant recombinant 10-FTHFDH are tetramers of the 99-kDa
monomers(4) .Enzymatic Activity of Mutant 10-FTHFDH
The
rationale for performing the site-directed mutagenesis was to confirm
the putative role of Cys-707 in the catalysis of the dehydrogenase
reaction. Assay of the C707A mutant in the presence of NADP showed no
dehydrogenase activity with either 10-HCO-HPteGlu or
10-FDDF (Fig. 2). The wild type recombinant enzyme assayed under
the same conditions showed dehydrogenase activity comparable to the
native rat liver enzyme.
PteGlu used as a substrate; B, 10-FDDF
used as a substrate.
of 638 µM and an
estimated V
of 245 nmol of NADP min mg
. These parameter were similar to those of
the natural rat liver enzyme. (
)The C707A mutant enzyme was
unable to oxidize propanal. These results show that the C707A mutant
10-FTHFDH has completely lost dehydrogenase activity. showed that the C707A mutant was able to catalyze the hydrolase
reaction. Hydrolase activity of the C707A mutant 10-FTHFDH was
comparable to the activity of wild type recombinant enzyme for both
10-HCO-H
PteGlu and 10-FDDF (Table 1).
Effect of NADP
To check whether the mutation influenced the
binding of the coenzyme, we titrated the quenching of tryptophan
fluorescence with NADP on 10-FTHFDH
Fluorescence
. No significant difference in
fluorescence quenching between wild type and C707A mutant recombinant
10-FTHFDH was observed (Fig. 3). The K
for
NADP binding calculated from these data were
approximately 0.3 µM for both wild type and mutant enzyme.
. Curves 1 (opencircles), wild type recombinant 10-FTHFDH; curves2 (darkcircles), C707A mutant
10-FTHFDH. Insertion shows the fluorescence data plotted in linear
form. The value (1 - F/F
)
was
plotted against the inverse of NADP
concentration(25) . This is a modified form of the
classical Stern-Volmer plot, which relates the drop in fluorescence to
the concentration of a collisional quencher (see (26) for
review). F is intrinsic fluorescence observed at an
NADP
concentration; F
is
fluorescence in the absence of NADP
. The slope of the
line (least squares fit) gave a K
that
was approximately 0.3 µM for both wild type and the C707A
mutant 10-FTHFDH. The protein was excited at 291 nm and the emission
monitored at 340 nm. 9.0 nM of each enzyme was used for the
analysis, and concentration of NADP
was varied from
0.02 µM to 10.0 µM. Variation of the measured
values was about 5%.
PteGlu and 10-FDDF. The C707A
mutant also lost the ability to oxidize propanal, while wild type
recombinant 10-FTHFDH oxidized propanal as well as the native rat liver
enzyme. Measurement of binding of the coenzyme, NADP,
by fluorescence titration showed no difference between the mutant and
wild type enzyme, indicating that loss of dehydrogenase activity was
not due to any change in the coenzyme binding site.
values for
NADP were similar for both enzymes. The behavior of
the mutant during the purification procedure was also identical to that
of the wild type recombinant protein, indicating no gross alteration of
the native structure(33) . It is known that native 10-FTHFDH
forms oligomers(2, 3, 16) ; therefore, we
also determined the oligomeric structure of the wild type recombinant
and the mutant enzymes. For this purpose, we used a laser-light
scattering technique. This technique involves analysis of the temporal
fluctuations in the intensity of light scattered by the Brownian motion
of macromolecules. It provides a rapid, accurate, and noninvasive
method of determining the translational diffusion coefficient of
macromolecules that can be related to an effective size(18) .
The analysis revealed that both wild type and mutant proteins existed
as tetramers. The fact that the entire population of the active wild
type recombinant 10-FTHFDH was organized as a tetramer indicates that
this is the functional form of the enzyme. Since the C707A mutant
10-FTHFDH also forms a tetramer, this suggests they have similar
conformations. Based on all these observations, we assume that the
substitution of an alanine for the cysteine did not change protein
conformation and stability. However, conservative mutations that
dramatically reduce activity without changes in protein stability
strongly suggest that a residue is important for substrate recognition
or activity.
)PteGlu,
10-formyltetrahydrofolate; HPteGlu, tetrahydrofolate;
10-FDDF, 10-formyl-5,8-dideazafolate; 2-ME, 2-mercaptoethanol; PCR,
polymerase chain reaction.)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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