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Journal of Bacteriology, February 1999, p. 718-725, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification and Properties of NADH-Dependent
5,10-Methylenetetrahydrofolate Reductase (MetF) from
Escherichia coli
Christal A.
Sheppard,
Elizabeth E.
Trimmer, and
Rowena G.
Matthews*
Biophysics Research Division and Department
of Biological Chemistry, The University of Michigan, Ann Arbor,
Michigan 48109-1055
Received 9 October 1998/Accepted 9 November 1998
 |
ABSTRACT |
A K-12 strain of Escherichia coli that overproduces
methylenetetrahydrofolate reductase (MetF) has been constructed, and
the enzyme has been purified to apparent homogeneity. A plasmid
specifying MetF with six histidine residues added to the C terminus has
been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine
dinucleotide (FAD). No additional cofactors or metals have been
detected. The purified enzyme catalyzes the reduction of
methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the
reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the Km for
NADH is 13 µM, the Km for
CH2-H4folate is 0.8 µM, and the turnover
number under Vmax conditions estimated for the
reaction is 1,800 mol of NADH oxidized min
1 (mol of
enzyme-bound FAD)1. NADPH also serves as a reductant, but
exhibits a much higher Km. MetF also catalyzes
the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in
the presence of menadione, which serves as an electron acceptor. The
properties of MetF from E. coli differ from those of the
ferredoxin-dependent methylenetetrahydrofolate reductase isolated from
the homoacetogen Clostridium formicoaceticum and more
closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent
enzymes from eukaryotes.
| |
INTRODUCTION |
In Escherichia coli,
methylenetetrahydrofolate reductase (MetF) catalyzes the reduction of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. This
reaction commits tetrahydrofolate-bound one-carbon units to use in the
methylation of homocysteine to form methionine, the terminal step in
methionine biosynthesis. Hatch et al. (14) first identified
the enzyme activity in crude extracts of E. coli. NADH was
shown to be more effective than NADPH as the source of reducing
equivalents in relatively crude preparations (4). During
purification of methylenetetrahydrofolate reductase from cell extracts,
the ability of the enzyme to be reduced by NADH was lost, necessitating
assay of the enzyme in the presence of NADH, an NADH-flavine adenine
dinucleotide (FAD) oxidoreductase, and FAD (17). Thus, it
was believed that catalysis of the overall reaction shown in equation 1 required two enzymes: a methylenetetrahydrofolate reductase that
catalyzed transfer of reducing equivalents from reduced FAD to
CH2-H4folate, and an NADH-FAD oxidoreductase
that catalyzed transfer of reducing equivalents from NADH to FAD.
|
(1)
|
The metF gene specifies methylenetetrahydrofolate
reductase. This gene is located in the metJBLF gene cluster,
which was cloned and mapped by Zakin et al. (35). The
metF gene was sequenced, and the gene product was shown to
be a polypeptide of 33 kDa (24). However, we are unaware of
publications reporting further characterization of the E. coli enzyme.
Methylenetetrahydrofolate reductase has previously been purified from
porcine liver (7) and has been shown to contain
noncovalently bound FAD and to use NADPH as a reductant. By using
peptide sequences from the porcine enzyme to design oligomers, a clone
for the human MTHFR gene was identified and sequenced, and
the catalytic domain of the human enzyme was shown to exhibit extensive
sequence similarity with MetF from E. coli (12).
While two other bacterial methylenetetrahydrofolate reductases have
been purified and characterized, their sequences have not been reported
and they appear to differ appreciably from the human and E. coli enzymes. The enzyme from Clostridium
formicoaceticum (5) is an iron-sulfur flavoprotein that
catalyzes reduction of CH2-H4folate with
reduced ferredoxin as an electron donor. Methylenetetrahydrofolate
reductase from Peptostreptococcus productus more closely
resembles the porcine and E. coli enzymes in that it lacks
iron and catalyzes the reduction of methylenetetrahydrofolate with NADH
as the electron donor (34). However, this enzyme appears to
be associated with the cell membrane, in contrast to the E. coli and mammalian enzymes.
In this paper, we report the construction of E. coli strains
for overproduction of methylenetetrahydrofolate reductase and the
purification and characterization of the enzyme. The E. coli enzyme serves as a useful model for the human enzyme, mutations of
which have been implicated in hyperhomocysteinemia in humans and in
risk for the development of cardiovascular disease (10, 16)
and neural tube defects (29). In work to be reported
elsewhere, the X-ray structure of MetF from E. coli has been
determined (13), and its availability will permit comparison
with F420-dependent enzymes with similar functions in
archaebacteria (22, 31). The ease of genetic manipulation of
the E. coli metF gene will also permit further studies to
define the functional properties of this important enzyme.
We have purified the MetF protein to homogeneity and shown it to be a
flavoprotein capable of catalyzing the NADH-linked reduction of
CH2-H4folate to
CH3-H4folate without added enzymes or
cofactors. The purified enzyme is a tetramer, but when diluted it
readily loses its FAD cofactor, possibly accounting for the loss of
catalytic function during earlier attempts at purification.
| |
MATERIALS AND METHODS |
Reagents.
Restriction enzymes and DNA-modifying enzymes were
obtained from Promega, New England BioLabs, or Boehringer Mannheim.
Taq polymerase and buffer were obtained from Boehringer
Mannheim. Vent polymerase and buffer were purchased from New England
BioLabs. Nonradioactive
(6R,S)CH3-H4folate and
H4folate were obtained from B. Schircks, Jona, Switzerland.
NADH, menadione, FAD, and dimedone were purchased from Sigma.
(6R,S)[methyl-14C]CH3-H4folate
was purchased from Amersham Life Sciences as the barium salt. Luria
broth was prepared as previously described (25).
Formaldehyde, formic acid, hydrochloric acid, methanol, ascorbic acid,
acrylamide, ampicillin, riboflavin, IPTG
(isopropyl-
-D-thiogalactopyranoside), EDTA, phosphate,
sodium chloride, glycerol, bovine serum albumin, nickel sulfate,
imidazole, phosphate, NaCl, glycerol, and agarose were supplied by
various commercial sources and were used without further purification.
HiTrap chelating columns were obtained from Pharmacia, Piscataway, N.J.
The DNA primers used for isolation of the metF gene were
synthesized at the University of Michigan DNA Synthesis Core Facility.
Assays. (i) CH3-H4folate-menadione
oxidoreductase assay.
In the presence of a high potential electron
acceptor like menadione, methylenetetrahydrofolate reductase oxidizes
[methyl-14C]CH3-H4folate
to 14CH2O (formaldehyde) and
H4folate (8, 19). Radiolabeled formaldehyde was
complexed with dimedone and extracted with toluene. Formation of the
formaldehyde product was measured by scintillation counting.
The 800-µl reaction mixture contained 60 µl of the radiolabeled
(6
R,
S)[
methyl-
14C]CH
3-H
4folate
cocktail (4.17 mM, 1,200 dpm/nmol), 100 µl of 1
M phosphate buffer
(pH 6.3), 60 µl of 0.5 mM FAD, 200 µl of menadione
stock, and 100 µl of 2% bovine serum albumin in 30 mM EDTA. This
mixture was
preincubated at 37°C for 5 min, and then enzyme was
added to initiate
the reaction. After 30 s, the assay was terminated
by the addition
of 300 µl of dimedone and heat treatment at 98°C
for 2 min. The
reaction mixtures were cooled on ice for 2 min,
and 3 ml of toluene was
added. The mixture was vortexed and spun
in a clinical centrifuge at
4,000 rpm for 5 min. A 1-ml volume
of the toluene phase was added to 10 ml of Eco-lite scintillation
cocktail (ICN Biomedicals Inc.) and
counted in a Beckman scintillation
counter. Units for this assay are
defined as micromoles of formaldehyde
formed per
minute.
The radioactive methyltetrahydrofolate cocktail for this assay was
prepared by adding 50 µCi of
5-[
14C](6
R,
S)methyltetrahydrofolic acid
(specific activity, 54 mCi/mmol)
to 5 ml of 8 mM ascorbic acid and
combining the mixture with 19
ml of ascorbate buffer containing 60.1 mg
of unlabeled (6
R,
S)CH
3-H
4folate.
A
menadione stock solution was prepared by diluting 1 volume of
a
saturated solution of menadione in methanol with 4 volumes of
water.
The precipitate that formed was removed by filtering. The
solution was
stirred in the dark for 10 min and then refiltered
and stored in the
dark. The concentration of menadione in this
stock solution was found
to be 1.4 mM by measuring
A340 and using
a molar
absorbance coefficient of 3,100 M
1 cm
1
(
30).
(ii) NAD(P)H-menadione oxidoreductase assay.
The
NAD(P)H-menadione oxidoreductase activity was measured at 25°C, using
the method of Donaldson and Keresztesy (8) as modified by
Matthews (19). In a total volume of 3 ml, the reaction mixture consisted of 1.5 ml of 100 mM potassium phosphate buffer (pH
7.2) with 0.6 mM EDTA, 300 µl of menadione stock (prepared as
described above), and 125 µM NADH. The mixture was incubated at
25°C for 5 min, and then enzyme was added to initiate the reaction. Activity was monitored as a decrease in the absorbance of NADH at 343 nm, where menadione and menadiol are isosbestic. Activities are
presented as the initial rate of NADH oxidation observed, using an
extinction coefficient of 6,220 M1 cm1 for
NADH at 343 nm. Activity units represent micromoles of NADH oxidized
per minute.
(iii) NADH-CH2-H4folate oxidoreductase
assay.
In the NADH-CH2-H4folate
oxidoreductase assay, MetF oxidizes NADH and reduces
CH2-H4folate to
CH3-H4folate. The assay is a modification of
the NADPH-CH2-H4folate oxidoreductase assay
originated by Kutzbach and Stokstad (18) and described by
Matthews (19). In our modification, the assay was performed
in a stopped-flow spectrophotometer at 15°C. MetF (20 µM in
enzyme-bound flavin) was mixed with an equal volume of buffer
containing NADH and CH2-H4folate. Cycles of
alternating vacuum and argon gas flow over the samples prior to mixing
were used to deaerate both solutions.
To determine the
Km and
KiA for NADH, deaerated 20 µM enzyme was mixed
anaerobically with deaerated solutions containing 60
µM
(6
R)CH
2-H
4folate and concentrations
of NADH varying from 10
to 400 µM. Enzyme activity was measured as a
decrease in the FAD
absorbance of the enzyme by monitoring the enzyme
absorbance at
447 nm. The buffer for both the enzyme and substrate
solutions
was 50 mM potassium phosphate (pH 7.2) (KP
i)
buffer containing
10% glycerol and 0.3 mM EDTA. The
CH
2-H
4folate was added as a
6
R,
S
mixture and was prepared anaerobically by adding 5 ml deaerated
50 mM
KP
i buffer, containing 10% glycerol and 0.3 mM EDTA, to
27.7 mg of H
4folate under nitrogen. Reagent formaldehyde
(81 µl
of a 14.26 M stock) was added to generate 10 mM
(6
R,
S)CH
2-H
4folate.
The
Km and
KiB for
CH
2-H
4folate were also determined by the NADH
CH
2-H
4folate oxidoreductase assay. Deaerated 20 µM enzyme
was mixed anaerobically with deaerated solutions containing
200
µM NADH and concentrations of
(6
R)CH
2-H
4folate varying from 30
to
2,000 µM. The oxidation of NADH was observed as a decrease
in the
absorbance at 340
nm.
Subcloning of metF.
Plasmid pEJ3-1B contains the
metF gene, including the 5' upstream region regulated by an
S-adenosylmethionine (AdoMet)-MetJ complex, half of
Tn5 including the gene coding for neomycin
phosphotransferase, and 1.5 kb of additional bacterial DNA 3' to
E. coli metF (9). To overproduce the desired
protein, extraneous material from the pEJ3-1B clone was removed and the
coding region of metF was subcloned into the
EcoRI site of plasmid pTrc99A (Pharmacia) to
generate the overexpression plasmid pCAS-5. The metF coding
region in pEJ3-1B was amplified by PCR using pEJ3-1B as a template and
primers 144C and 145C*, where the asterisk denotes a sequence
corresponding to the reverse complement of the sense strand. Primers
144C
(5'-ggaactgcagATTGATGAGGTAAGGTATGA--3') and 145C*
(5'-ggaagacgtcTTATAAACCAGGTCGAACCCC-3') were
generated to anneal to the 5' and 3' ends, respectively, of the
E. coli metF coding sequence. The primers were designed to
include the metF ribosome binding site, GAGG,
the ATG translation initiation codon at the 5' end of the
gene, and a sequence complementary to the TAA stop codon,
TTA, at the 3' end of the gene. In addition, primers 144C
and 145C* contain 10 bp of DNA sequence (lowercase) that are not
contained within the metF gene but appear in the final
construct at the 5' and 3' ends of the metF gene.
The 927-bp PCR product was composed of 888 bp comprising the coding
region of
metF, the 3-bp termination codon, 16 bp of 5'
untranslated region that contained the ribosome binding site,
and 20 bp
introduced by the primers that were not part of
metF.
Ligation of the PCR product into the Invitrogen TA cloning vector
pCRII
generated plasmid pCAS-3. This plasmid was then digested
with the
restriction enzyme
EcoRI to generate a 943-bp fragment.
The
EcoRI site was contained within plasmid pCRII, adding 16 bp
from this plasmid to the 927-bp PCR fragment containing the
metF gene. This 943-bp fragment was ligated into the
EcoRI site of
dephosphorylated expression vector
p
Trc99A (Pharmacia) to generate
plasmid pCAS-5.
Purification of wild-type MetF.
E. coli AB1909
(metF arg lac), which contains a mutation in metF
that leads to methionine auxotrophy (32), was transformed with pCAS-5 to generate the overexpression strain CAS-9. Cells of
strain CAS-9 were inoculated in LB to an optical density at 420 nm
(OD420) of 0.05, grown aerobically in a rotary action
shaker at 37°C to an OD420 of 1.0, and then induced with
0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) and
allowed to continue growing for 3 h. The cells were pelleted and
resuspended in 50 mM KPi buffer (pH 7.2) containing 10%
glycerol and 0.3 mM EDTA. The protease inhibitors phenylmethylsulfonyl fluoride and tosyl-L-lysine chloromethylketone were added
to final concentrations of 50 µM and 100 µM, respectively, prior to
sonication. The cells were sonicated with a Branson sonifier at a
setting of 8. To prevent overheating, seven 1-min sonication cycles
were alternated with 2-min recovery periods. The sonicate was
centrifuged at 35,000 × g for 1 h at 4°C, and
the supernatant was collected.
Purification of MetF to homogeneity required a three-column
purification process involving anion-exchange and hydrophobic
interaction chromatography. It is similar to the procedure developed
by
Wohlfarth et al. (
34) for the purification of
methylenetetrahydrofolate
reductase from
P. productus. DEAE
fast-flow Sepharose resin was
used for the first separation. The
sonicate supernatant was applied
to a 2- by 50-cm DEAE column
equilibrated with 50 mM KP
i containing
10% glycerol and
0.3 mM EDTA. The column was washed with 50 mM
KP
i-10%
glycerol-0.3 mM EDTA and eluted with a linear gradient
from 50 to 500 mM KP
i. The fraction containing MetF can be identified
by
absorbance at 447 nm; if sufficient enzyme is present, MetF
can be
visually identified by its intense yellow color. As this
step is a bulk
purification to remove DNA, RNA, and cellular debris,
the yellow
fractions were combined without regard to enzyme purity.
For buffer
exchange of the pooled protein fractions, an Amicon
apparatus with a
YM100 membrane was used. Care was taken to prevent
dilution of the
protein below 100 µM, which results in FAD dissociation
and protein
precipitation. Concentrating the protein solution
and then adding
buffer to restore the volume prior to concentration
accomplished buffer
exchange. The procedure was repeated until
the protein was in 50 mM
KP
i (pH 7.2) with 10% glycerol and 0.3
mM
EDTA.
The second purification step used fast protein liquid chromatography
(FPLC) with a Pharmacia Mono Q anion-exchange resin.
A Mono Q HR 16/10
column (20-ml bed volume) was used, and MetF
was eluted with a salt
gradient from 50 to 375 mM KP
i with 10%
glycerol and 0.3 mM EDTA. MetF elutes at approximately 300 mM
KP
i. The
cleanest fractions, as determined by electrophoresis
on sodium dodecyl
sulfate (SDS)-polyacrylamide gels, were combined,
and 1 volume of 1.6 M
(NH
4)
2SO
4 was added so that the
final buffer
composition of the protein was approximately 0.8 M
(NH
4)
2SO
4-100
mM KP
i
(pH 7.2) with 5% glycerol and 0.15 mM EDTA. For the third
purification
step, the protein was then loaded onto a 2- by 10-cm
Phenyl-sepharose
HP FPLC column (Pharmacia) preequilibrated with
0.8 M
(NH
4)
2SO
4 in 100 mM KP
i
(pH 7.2) containing 10% glycerol
and 0.3 mM EDTA. A reverse gradient
from 0.8 to 0.0 M (NH
4)
2SO
4 in the
same buffer was used to elute the enzyme. Pure
E. coli MetF
eluted at the very end of the gradient. The protein was exchanged
into
50 mM KP
i (pH 7.2) with 10% glycerol and 0.3 mM EDTA by
size
exclusion chromatography and stored at
80°C at a minimum
concentration
of 150 µM to ensure long-term protein
stability.
Protein concentration determinations.
Protein determinations
were performed by a Coomassie blue dye-binding method (3)
implemented via the Bio-Rad protein assay (Hercules, Calif.) and were
compared with standard curves obtained with bovine serum albumin.
Determination of the protein concentration of purified MetF by amino
acid analysis gave a value 65.2% of that obtained by the Bio-Rad
protein assay. Therefore, a factor of 0.652 was used to correct values
obtained by the Bio-Rad protein assay for samples of enzyme with
specific activities of >2.4 µmol min1
mg1 in the CH3-H4folate-menadione
oxidoreductase assay. Enzyme concentrations were routinely determined
from the visible absorbance at 447 nm due to bound FAD, using a molar
absorbance coefficient of 14,300 M1 cm1
(see Results).
Amino acid analysis.
Analyses were performed at the
University of Michigan Protein and Carbohydrate Core Facility, using an
Applied Biosystems model 420H automated amino acid analyzer, with
precolumn derivatization of the protein hydrolysate with phenylisothiocyanate.
Construction of a strain producing histidine-tagged MetF.
The PCR-based primer overlap extension method (15) was used
to produce a plasmid for production of histidine-tagged MetF. Generation of the histidine-tagged version of MetF required the removal
of the stop codon from the metF gene and insertion of the
coding sequence for the gene into the pET-23b vector
(Novagen, Milwaukee, Wis.), which contains six histidine codons and a
termination codon downstream of and in frame with a 6-bp
XhoI site. Primers 144C (see above), 3311E*
(5'-cccagtgctgcaatgataccg-3'), 3312E* (5'-gcttgcatgcctgcactcGAGTAAACCAGGTCGAAC-3'),
and 3313E
(5'-GTTCGACCTGGTTTACTCgagtgcaggcatgcaagc-3') were used to generate plasmid pCAS-28 containing the wild-type coding sequence with the original stop codon removed. The asterisks indicate sequences corresponding to the reverse complement of the sense
strand. Sequences contained within the coding sequence are shown in
uppercase, while those from flanking regions are shown in lowercase;
underlined sequences indicate changes made to introduce an
XhoI site in place of the original stop codon. Separate
amplification reactions were performed with the wild-type expression
plasmid pCAS-5 as the DNA template and either 144C and 3312E* or 3313E
and 3311E* as primers. The two amplified DNA fragments were then
combined and amplified by using 144C and 3311E* as primers. The
resultant 2,245-bp PCR product was digested with BglI and
BspEI to generate a 1,696-bp fragment, containing the entire
E. coli metF gene with a XhoI site instead of a
stop codon at the 3' end of the gene. This fragment was reintroduced
between the BglI and BspEI sites in pCAS-5,
replacing the original metF sequence with one that contained
an XhoI restriction site at the carboxy terminus and no stop
codon. The resultant plasmid, pCAS-28, was digested with
XbaI and XhoI to generate a 922-bp fragment containing the full-length mutated gene. Plasmid pET-23b was
digested with XbaI and XhoI, which removed a
117-bp fragment from the plasmid that specifies an unwanted
amino-terminal T7 tag fusion protein, the vector-encoded ribosome
binding site, and part of the multicloning region. The 922-bp
XbaI-XhoI gene fragment was ligated into the 3,547-bp XbaI-XhoI fragment of pET-23b
to generate the expression vector pCAS-30. This plasmid contains the
metF coding sequence juxtaposed to a C-terminal histidine
tag. The resulting construct specifies a protein that contains two
mutated residues (Leu-Glu) at the C terminus of the original sequence,
introduced by formation of the XhoI site, followed by six
histidine residues.
Production of histidine-tagged MetF.
The histidine-tagged
protein was produced by using E. coli EET01, which contains
plasmid pCAS-30 in a BL21(DE3)recD+ (Novagen)
background. Use of the BL21(DE3)recD strain results in
substantially lower levels of accumulation. The BL21(DE3) background contains a chromosomal copy of the T7 RNA polymerase under control of
the lacUV5 promoter. T7 RNA polymerase was required for
production of the histidine-tagged proteins. Although polymerase
production is induced by IPTG in this strain, we did not observe
increased accumulation of MetF on addition of IPTG, and IPTG was not
added to any of our cultures.
One liter of Luria broth, supplemented with ampicillin (100 µg/ml)
and riboflavin (10 µM), was inoculated to an OD
600 of
0.015
with strain EET01. The cells were allowed to grow in a rotary
action shaker at 37°C until they reached stationary phase
(OD
600 of ~5), after approximately 9 h. The cells
were pelleted at 11,000
×
g for 10 min, and the
supernatant was
removed.
Purification of histidine-tagged methylenetetrahydrofolate
reductase.
Pelleted cells producing histidine-tagged MetF were
resuspended by adding 50 µl of ice-cold buffer (10 mM imidazole in 20 mM phosphate buffer [pH 7.4] containing 500 mM NaCl) per ml of cell
culture. The cell suspensions were then sonicated in the presence of
the protease inhibitors phenylmethylsulfonyl fluoride and
tosyl-L-lysine chloromethyl ketone as described above. The supernatant was separated from cell debris by centrifugation at 35,000 × g for 1 h at 4°C. Purification of the
histidine-tagged protein was accomplished by using a nickel affinity
column. The nickel column was generated by charging the HiTrap
chelating column (Pharmacia) with Ni2+, using nickel
sulfate. Using a peristaltic P-1 pump (Pharmacia), the sonicate
supernatant was loaded onto a 5-ml nickel column previously
equilibrated with 50 ml of 10 mM imidazole in 20 mM phosphate (pH 7.4)
with 500 mM NaCl. The histidine-tagged protein bound to the nickel
column along with some nonspecifically bound protein. Nonspecifically
bound proteins were eluted by washing with 50 ml of loading buffer
followed by 50 ml of the same buffer containing 100 mM imidazole.
Homogeneous histidine-tagged E. coli MetF was then eluted
with the same buffer containing 300 mM imidazole. Fractions (400 µl)
were collected, and glycerol was added to 10% (wt/vol). Approximately
4 µg of protein from each fraction was run on an SDS-polyacrylamide
gel to identify pure fractions. The pure fractions were pooled, and the
buffer was exchanged to 50 mM KPi, (pH 7.2) with 10%
glycerol and 0.3 mM EDTA, using size exclusion chromatography on an
FPLC Superose-12 column. The protein was stored at 80°C at a
concentration of >100 µM.
Determination of the molar absorbance of enzyme-bound FAD.
Enzyme-bound flavin was released by denaturing the enzyme by addition
of an equal volume of 7.5 M guanidine HCl in 1 M Tris-HCl at pH 7.2. The absorbance of the liberated FAD was compared to the absorbance of a
known quantity of free FAD. The molar absorbance coefficient of FAD in
aqueous solution at neutral pH is 11,300 M1
cm1 at 450 nm (1); the molar absorbance
increases 4.85% on dilution with guanidine hydrochloride
(6), yielding a molar absorbance coefficient of 11,850 M1 cm1.
Apparent mass of the native enzyme.
The oligomeric state of
the MetF protein was determined by size exclusion chromatography using
an FPLC Superose-12 HR 10/30 gel filtration column (Pharmacia).
Concentrated protein was diluted to 61 µM in 50 mM KPi
buffer (pH 7.2) with 10% glycerol and 0.3 mM EDTA. The protein was
applied to the column and eluted with 50 mM KPi (pH 7.2)
with 10% glycerol and 0.3 mM EDTA at 0.5 ml/min. The migration and
elution of the protein peak was ascertained by continuous monitoring of
the column eluant at 276 nm. The elution volume of the protein was
compared to the elution volumes of gel filtration protein standards
(Bio-Rad) dissolved in the same buffer. The elution volumes of the
standards were used to generate a calibration curve for the column, and
the native molecular weight for the MetF samples was calculated using
this calibration curve. The subunit molecular weight of
histidine-tagged MetF was determined by electrospray mass spectrometry
at the University of Michigan Protein and Carbohydrate Core Facility.
| |
RESULTS |
Subcloning of metF and construction of an overproducing
strain.
Plasmid pEJ3-1B, containing the E. coli metF
gene specifying methylenetetrahydrofolate reductase, was obtained as a
generous gift from James Johnson (9). This plasmid also
contains the 5' upstream region regulated by an AdoMet-MetJ complex,
half of Tn5 including the gene coding for neomycin
phosphotransferase, and 1.5 kb of additional bacterial DNA 3' of
E. coli metF. To overproduce MetF, it was desirable to
remove the regulatory region, so that cells could be grown in LB medium
without down-regulation of metF expression. Extraneous
material from the pEJ3-1B clone was removed as described in Materials
and Methods, and the coding region of metF was subcloned
into pTrc99A to generate the overexpression plasmid pCAS-5.
This plasmid contains a 943-bp DNA fragment of which 907 are bp 4151 to
5057 of metF (numbered according to GenBank accession no.
AE000468) cloned into the EcoRI site of pTrc99A. Plasmid pCAS-5 was introduced into E. coli AB1909
(metF) to create strain CAS-9. Following growth in LB
containing IPTG, approximately 20% of the total cellular protein is
E. coli MetF. Thus, removing the regulatory region and
placing protein production under the control of the trc
promoter leads to high levels of MetF accumulation.
Purification of MetF to homogeneity.
Wild-type E. coli MetF enzyme was produced from strain CAS-9 as described in
Materials and Methods. Purification of MetF to homogeneity requires a
three-column purification process involving anion-exchange and
hydrophobic interaction chromatography (see Materials and Methods for
details). Table 1 summarizes the
purification and reports specific activities measured with the
CH3-H4folate-menadione oxidoreductase assay.
Precautions are taken such that the enzyme is not diluted to below 100 µM to eliminate dilution-related inactivation of the protein. In
particular, size exclusion chromatography was used for buffer exchange
prior to storage of homogeneous enzyme. The protein was stored at
80°C in a solution of 50 mM KPi (pH 7.2) with 10%
glycerol and 0.3 mM EDTA at a minimum concentration of 150 µM to
ensure long-term protein stability. Protein is stable at this
concentration for up to a year, provided that it does not undergo
repeated freezing and thawing.
Properties of purified methylenetetrahydrofolate reductase.
The wild-type MetF protein is isolated as a flavoprotein as indicated
by its yellow color. The flavin is nonfluorescent when bound to MetF
apoenzyme but becomes fluorescent when released from the protein, e.g.,
by thermal denaturation. Since FAD is much less fluorescent than flavin
mononucleotide (FMN), the two forms of flavin can be distinguished by
treatment of the released flavin with snake venom phosphodiesterase,
which converts FAD to FMN with an accompanying 10-fold increase in
fluorescence (2). In this manner, the flavin bound to MetF
was shown to be FAD, as suggested from earlier studies (14,
17). The molar absorbance of MetF was determined by comparing the
absorbances of the enzyme before and after the release of FAD. FAD was
released by denaturing the enzyme with guanidine hydrochloride and
comparison of the absorbance of the liberated FAD with that of a known
quantity of free FAD (1). The molar extinction coefficient
was determined to be 14,300 M1 cm1 for the
enzyme-bound FAD. The enzyme concentration that was determined from the
absorbance at 447 nm due to enzyme-bound FAD was 1.06 times the subunit
concentration determined by amino acid analysis, indicating that within
experimental error, the enzyme is isolated with one equivalent of FAD
bound per subunit.
The enzyme was found to lose activity upon dilution. As is evident from
the data in Table
1, significant losses were observed
during
purification, especially during chromatographic procedures
that
resulted in dilution of the enzyme. We therefore decided
to construct a
modified MetF with a histidine tag at the C terminus,
so that the
protein could be purified by chromatography on nickel
agarose. Strain
BL21(DE3) was transformed with pCAS-30 (see Materials
and Methods) and
used to produce the histidine-tagged MetF protein.
A summary of the
purification is shown in Table
2. The
protein
could be purified to homogeneity in a single step with 96%
yield.
The specific activity of the purified protein (5.37 U/mg) is
comparable
to that of the wild-type protein (4.67 U/mg), but 1 liter of
cell
culture yielded 58 mg of purified MetF, compared to 4 mg obtained
per liter of cells producing wild-type MetF.
Histidine-tagged MetF protein has a predicted subunit molecular weight
of 34,062, calculated assuming that the N-terminal
methionine is
removed. Determination of a peptide mass of 34,072
± 10 by
electrospray mass spectrometry was consistent with removal
of the
initiator methionine residue. The oligomeric state of the
histidine-tagged MetF protein was determined by size exclusion
chromatography as described in Materials and Methods. The elution
volume of MetF is compared with those of standard proteins in
Fig.
1. MetF applied to the column at an
initial concentration
of 61 µM enzyme-bound FAD eluted at a volume
equivalent to a molecular
mass of 119 ± 2 kDa, or 3.5 subunits of
34 kDa per molecule, suggesting
that the protein is a tetramer. The
recent determination of the
structure of
E. coli MetF by
X-ray crystallography confirms the
tetrameric structure of the native
wild-type (non-histidine-tagged)
enzyme (
13).
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|
FIG. 1.
Determination of the apparent molecular weight of
histidine-tagged MetF by gel filtration. The oligomeric state of MetF
was determined by size exclusion FPLC on a Superose-12 HR 10/30 gel
filtration column. Concentrated protein was diluted to 61 µM in 50 mM
KPi (pH 7.2) containing 10% glycerol and 0.3 mM EDTA,
loaded on the column, and eluted with the same buffer. The elution
volume is compared with those of compounds in the Bio-Rad gel
filtration standard kit. These compounds and their molecular weights
are as follows (unfilled circles, from left to right): bovine
thyroglobulin, 670,000; bovine gamma globulin, 158,000; chicken
ovalbumin, 44,000; horse myoglobin, 17,000; and vitamin
B12, 1,350. The results of duplicate determinations of the
standard curve are indicated. The duplicate determinations of MetF
(filled circle) yielded identical elution volumes corresponding to an
apparent molecular weight of 119,400. This molecular weight is
consistent with the concentrated native enzyme eluting as a tetramer of
expected Mr of 136,248 (4 × 34,062).
|
|
Histidine tagging did not affect any of the enzyme properties that we
have examined. The unmodified enzyme also migrates as
a tetramer during
size exclusion chromatography. Differential
scanning calorimetry
indicated similar melting temperatures for
the wild-type (58.6°C) and
histidine-tagged (58.4°C) proteins.
Thus, the introduction of
additional residues at the C terminus
of the protein does not appear to
have altered the physical properties
of the
enzyme.
Catalytic properties of methylenetetrahydrofolate reductase.
Figure 2 shows the absorbance spectrum of
the purified wild-type enzyme. This spectrum, with maxima at 447 and
380 nm, is characteristic of a flavoprotein. On addition of NADH, the
spectrum is converted to a featureless curve lacking discrete bands
above 400 nm; this spectrum is characteristic of a reduced
flavoprotein, indicating that NADH is able to reduce the flavin of
MetF. The initial rate of reduction of MetF by various concentrations
of NADH was monitored in a stopped-flow spectrophotometer at 15°C. The results are shown in Fig. 3. A
saturable hyperbolic binding curve for NADH is observed, with an
apparent Kd of 37 µM and a maximum observed
rate of 34 s1, or ~2,000 min1.
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|
FIG. 2.
Overlay of oxidized and NADH-reduced MetF spectra.
Purified enzyme, 22 µM in potassium phosphate buffer (pH 7.2)
containing 10% glycerol and 0.3 mM EDTA, was equilibrated with argon,
and the spectrum was recorded. The absorbance shown (solid line) has
been corrected for the subsequent twofold dilution of the enzyme and is
equivalent to that of 11 µM enzyme. The enzyme was mixed with an
equal volume of 200 µM NADH in the same buffer, and the spectrum of
the reduced enzyme (dashed line) was recorded.
|
|
View larger version (13K):
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FIG. 3.
Reaction of MetF with NADH at 15°C. Under anaerobic
conditions, 20 µM oxidized enzyme in potassium phosphate buffer (pH
7.2) containing 10% glycerol and 0.3 mM EDTA was mixed with various
concentrations of NADH dissolved in the same buffer. The reaction was
monitored at 447 nm, where the absorbance of the oxidized enzyme-bound
FAD is maximal, and initial rates of reduction were determined for each
concentration. The corresponding rate constants
(kobx) are plotted against the NADH
concentration. The data were fit to a hyperbolic equation (solid line)
and yielded a Kd of 37 µM for NADH and a
maximal value for kobs of 34 s1.
|
|
The physiological reaction catalyzed by MetF is the reduction of
CH
2-H
4folate to form
CH
3-H
4folate. We monitored the physiological
reaction at 15°C in a stopped-flow spectrophotometer by mixing
enzyme
with CH
2-H
4folate and NADH. The enzyme and
substrate solutions
were equilibrated with argon prior to mixing, and
the reaction
was conducted under rigorously anaerobic conditions.
Details of
the procedure are presented in Materials and Methods.
Enzyme,
20 µM in bound FAD, was mixed with an equal volume of buffer
containing
60 µM (6
R)CH
2-H
4folate
and 10 to 400 µM NADH, and the initial
rate of flavin reduction was
monitored at 447 nm. The measured
initial velocity is plotted against
the NADH concentration. The
inset to Fig.
4 shows the initial rate of NADH
oxidation measured
at 340 nm when 20 µM enzyme is mixed with an equal
volume of buffer
containing 200 µM NADH and 30 to 2000 µM
(6
R)CH
2-H
4folate. Marked
inhibition
of the reaction is seen at high levels of
CH
2-H
4folate,
as is characteristic of enzymes
that follow ping-pong kinetic
mechanisms (
11). The
Vmax for this reaction must be corrected
for the
observed excess substrate inhibition; equation 2 has been
used to
determine the
Km values for NADH (
A)
and for CH
2-H
4folate
(
B), the
KiA and
KiB values for
substrate inhibition by NADH and
CH
2-H
4folate
respectively, and
Vmax (
26) by
successive iterations
using Kaleidograph (Abelbeck Software, Reading,
Pa.) and comparison
of the predicted curves with the data shown in the
graph and inset.
(2)
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|
FIG. 4.
Measurement of turnover in the
NADH-CH2-H4folate oxidoreductase reaction at
15°C. Under anaerobic conditions in a stopped-flow spectrophotometer,
enzyme, 20 µM in 50 mM potassium phosphate buffer (pH 7.2) containing
10% glycerol and 0.3 mM EDTA, was mixed with an equal volume of the
same buffer containing 30 µM
(6R)CH2-H4folate and various
concentrations of NADH. The initial velocity, measured as the rate of
reduction of the enzyme at 447 nm, is plotted against the NADH
concentration after mixing. The inset shows a plot of the initial
velocity, measured as the rate of formation of NADH at 340 nm,
determined when 20 µM enzyme was mixed with an equal volume of buffer
containing 200 µM NADH and various concentrations of
CH2-H4folate [added as a
(6R,S)CH2-H4folate mixture]. Marked
excess substrate inhibition is evident. Using equation 2, the
solid-line fits to the data were determined, yielding the following
values for kinetic parameters: KmA (NADH),
13 ± 2 µM; KiA, 9 ± 2 µM;
KmB
[(6R)CH2-H4folate], 0.8 ± 0.2 µM; KiB, 7.1 ± 1.4 µM; and
Vmax/ET, 30 ± 3 s1.
|
|
Using this equation,
Vmax/
ET was calculated to
be 30 ± 3 s
1,
KmA was
13 ± 2 µM,
KiA was 9 ± 2 µM,
KmB was 0.8 ± 0.2 µM, and
KiB was 7.1 ± 1.4 µM. A turnover number
of 1,800 min
1 can be calculated for this reaction under
Vmax conditions at
15°C.
An unexpected feature of the reaction catalyzed by MetF is that the
initial rate of reduction of the enzyme-bound flavin,
which takes place
during the approach to steady state, is the
same as the observed rate
of NADH oxidation during the steady
state. Note that in Fig.
4, the
initial rate of NADH oxidation
in the presence of 30 µM
CH
2-H
4folate is ~160 µM s
1
(
v/
ET is 16 s
1) whether the
reaction is monitored at 340 nm, where NADH absorbs
(inset), or at 447 nm, where FAD absorbs (main graph). This agreement
is seen only when
the overall rate of reaction is limited by the
rate of reduction of the
flavin, such that
kred <<
kox as defined
by equations 3 and 4 describing
the reductive and oxidative half
reactions:
|
(3)
|
|
(4)
|
In agreement with this conclusion, the turnover number, estimated
to be ~1,800 min
1 after correction for excess substrate
inhibition, is only slightly
lower than the rate constant for reduction
of the enzyme when
NADH is saturating, which is 2,000 min
1 as deduced from the data in Fig.
3.
In addition to the physiological reaction, methylenetetrahydrofolate
reductase also catalyzes transfer of reducing equivalents
from reduced
pyridine nucleotides to menadione, an artificial
electron acceptor, as
shown in equation 5:
|
(5)
|
The
Km for NADH was determined to be 30 µM in this assay at 25°C, while the
Km for
NADPH was >300 µM under the same conditions.
Thus, NADH rather than
NADPH appears to be the physiological source
of reducing equivalents
for the enzyme-bound flavin of MetF, as
previously deduced from studies
with crude extracts (
14).
Neither the physiological reaction nor the NADH-menadione
oxidoreductase reaction is suitable for assays of crude extracts;
a
CH
3-H
4folate-menadione oxidoreductase assay is
usually used
for these purposes (
18,
19). When this reaction
is measured
at 37°C, a
Km for
(6
S)CH
3-H
4folate of 75 µM is
observed, and the
turnover number under
Vmax
conditions is 190 min
1. Values approaching this turnover
number are obtained only with
assay incubation times of 30 s,
since plots of enzyme activity
versus time are linear for only 1
min.
The specific activities for the purified wild-type and histidine-tagged
proteins were identical within experimental error
for the
NADH-menadione oxidoreductase assay at 25°C and the
CH
3-H
4folate-menadione
oxidoreductase assay at
37°C.
| |
DISCUSSION |
Properties of our enzyme preparations compared to those for enzymes
from other prokaryotes.
Methylenetetrahydrofolate reductase from
E. coli differs significantly from the enzymes with similar
function previously purified from prokaryotes. The enzyme from C. formicoaceticum has been purified to homogeneity (5).
This enzyme is an 
44 octamer containing
26- and 35-kDa subunits. It contains 15 irons and 20 acid-labile
sulfurs per octamer of 237,000, and the spectrum is characteristic of a
flavoprotein containing iron-sulfur centers. The enzyme also contains
1.7 FAD per octamer and 2.3 zinc. The enzyme-bound FAD is not reduced
by NADH or NADPH, but it catalyzes the reduction of
CH2-H4folate with reduced ferredoxin as an
electron donor.
Wohlfarth et al. (
34) have purified
methylenetetrahydrofolate reductase from
P. productus to
homogeneity. This enzyme is
an
8 octamer of 32-kDa
subunits and contains 4 FAD per octamer.
About 40% of the enzyme is
recovered in the particulate fraction,
suggesting that the enzyme is
associated with the cell membrane.
In contrast to the clostridial
enzyme, this enzyme lacks iron
and catalyzes the reduction of
methylenetetrahydrofolate with
NADH as the electron donor. The enzyme
from
E. coli more closely
resembles the
Peptostreptococcus enzyme and eukaryotic
methylenetetrahydrofolate
reductases. However, it differs from these
enzymes by being an
4 tetramer of identical subunits,
each of which contains bound
FAD if the enzyme is isolated without
storage at dilute
concentrations.
It is difficult to compare the specific activities of the enzymes
from
P. productus and
E. coli. The specific
activity of
the
P. productus enzyme in the
NADH-CH
2-H
4folate assay was measured
at pH 5.5 and 37°C and found to be 380 µmol min
1 mg of
protein
1. The specific activity was reported to decrease
as the pH was
raised. In contrast, the specific activity of the
E. coli enzyme
in the same assay was measured at pH 7.2 and
15°C and found to
be ~1 µmol min
1 mg of
protein
1; the variation of specific activity with pH has
not yet been
determined.
Comparison of properties of the enzymes from E. coli
and porcine liver.
The porcine liver enzyme has been purified to
homogeneity (6) and extensively characterized. By using
peptide sequences obtained from the porcine enzyme, a cDNA specifying
human methylenetetrahydrofolate reductase has been cloned and sequenced
(10, 12). Subsequently, sequences have been obtained for
putative methylenetetrahydrofolate reductase proteins from
Saccharomyces cerevisiae (28), Arabidopsis thaliana (23), and Caenorhabditis elegans
(33). The eukaryotic proteins are much larger (70 to 77 kDa)
than the MetF protein from E. coli, and the N-terminal
halves of the proteins show extensive sequence similarity with MetF.
Limited proteolysis of the native porcine enzyme with trypsin cleaves
the 77-kDa polypeptide into an N-terminal fragment of ~40 kDa
and a C-terminal fragment of ~37 kDa. Cleavage does not affect
the activity of the enzyme but abolishes allosteric regulation of
activity by AdoMet (21). Photoaffinity labeling established
that AdoMet binds to the C-terminal 37-kDa domain (27).
The porcine enzyme is a dimer, but scanning transmission electron
microscopy of preparations of enzyme that have been shadowed
with
uranyl sulfate reveals the appearance of tetrameric planar
rosettes,
suggesting that each subunit consists of two separable
domains
(
21). Sequence comparisons identify the N-terminal domain
as
the catalytic domain, presumably responsible for binding of
FAD and
substrates, and the C-terminal domain is thought to be
involved in
allosteric regulation of enzyme activity. In contrast,
the bacterial
enzyme is a tetramer of identical subunits, also
arranged in a planar
rosette with only one twofold axis of symmetry
(
13).
Table
3 compares the properties of the
methylenetetrahydrofolate reductase enzymes from
E. coli and
porcine liver. Both the
prokaryotic and eukaryotic enzymes are capable
of transferring
reducing equivalents from reduced pyridine nucleotides
to either
CH
2-H
4folate or menadione, and they
can also transfer reducing
equivalents from
CH
3-H
4folate to menadione. As mentioned above,
the enzyme from
E. coli uses NADH in preference to NADPH,
while
the porcine enzyme uses NADPH preferentially. However, the
porcine
enzyme can use NADH as a reductant, provided that phosphate
ions
are present in the assay buffer (
20).
The enzyme from
E. coli is markedly less stable than
its eukaryotic counterpart and readily loses its activity and flavin
cofactor when present in dilute solution. In a study to be reported
elsewhere (
13), we have shown that activity loss on dilution
results when the tetramer dissociates into dimers and is associated
with release of the flavin cofactor. Addition of FAD to the buffer
does
not completely prevent dissociation of the tetramer into
dimers when
the enzyme is dilute, although it slows the net rate
of dissociation.
These properties pose difficulties both for purification
and for assay
and led us to construct a vector for overproduction
of a
histidine-tagged MetF protein that can be purified in a single
step and
maintained at high concentration throughout the
purification.
| |
ACKNOWLEDGMENTS |
This work was supported in part by NIH grant GM24908, by NIH
predoctoral fellowship DK49201 (C.A.S.), and by an NIH Cellular & Molecular Biology training grant to the University of Michigan (C.A.S.).
We thank Vincent Massey, David Ballou, and Bruce Palfey for help with
the performance and analysis of the stopped-flow experiments. Plasmid
pEJ3-1B was provided by James Johnson.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biophysics
Research Division, The University of Michigan, Ann Arbor, MI
48109-1055. Phone: (734) 764-9459. FAX: (734) 764-3323. E-mail:
rmatthew{at}umich.edu.
| |
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Journal of Bacteriology, February 1999, p. 718-725, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
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