(Received for publication, May 15, 1995; and in revised form, July 6, 1995)
From the
Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is
the first enzyme to be identified whose substrate is an organic radical
and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA
has been cloned from cucumber seedlings (Sano, S., and Asada, K.(1994) Plant Cell Physiol. 35, 425-437), and a plasmid was
constructed in the present study that allowed a high level expression
in Escherichia coli of the cDNA-encoding MDA reductase using
the T7 RNA polymerase expression system. The recombinant MDA reductase
was purified to a crystalline state, with a yield of over 20 mg/liter
of culture, and it exhibited spectroscopic properties of the FAD
similar to those of the enzyme purified from cucumber fruits during
redox reactions with NADH and MDA. The red semiquinone of the FAD of
MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the
enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the
reduced enzyme to MDA. The specificity of electron acceptors of the
recombinant enzyme appeared to be similar to that of MDA reductase,
even though the amino acid sequence encoded by the cDNA was somewhat
different from that of the enzyme purified from cucumber fruits. The K values for NADH and NADPH of the
recombinant enzyme indicated a high affinity of the enzyme for NADH.
The reaction catalyzed by the enzyme did not exhibit saturation
kinetics with MDA up to 3 µM. A second order rate constant
for the reduction of the enzyme-FAD with NADH was 1.25
10
M
s
, as determined
by a stopped-flow method, and its value decreased with increases in
ionic strength, an indication of the enhanced electrostatic guidance of
NADH to the enzyme-FAD.
Since the first isolation of ascorbate (AsA) ()from
Hungarian red pepper (Svirbery and Szent-Györgyi,
1933), it has been established that AsA functions as an antioxidant and
protects cells from oxidative stress. The level of AsA in plant cells,
as postulated from its initial isolation from plant tissues, is high as
compared to that in mammalian cells, and an AsA-specific peroxidase
scavenges hydrogen peroxide. In mammalian cells, by contrast, a
selenium-containing glutathione peroxidase plays a major role (Asada,
1992). When AsA acts as an antioxidant in cells, in most cases,
monodehydroascorbate radicals (MDA) are produced as the primary
oxidation product. AsA peroxidase in plants generates MDA when it
scavenges hydrogen peroxide (Hossain et al., 1984), as do
guaiacol peroxidases such as horseradish peroxidase (Yamazaki and
Piette, 1961). Superoxide and hydroxyl radicals oxidize AsA to MDA.
Other organic oxidizing radicals, such as tocopherol chromanoxy,
carbon-centered, aminooxy, peroxy, and phenoxy radicals, are generated
under oxidative stress and generate MDA via their interactions with AsA
(Bielski, 1982). The glutathione thiol radical is produced by the
interaction of GSH with various radicals (Winterbourn, 1993), and it
generates MDA as a result of its reaction with AsA (Forni et
al., 1983). Thus, the MDA radical functions as a
``sink'' for radicals and active species of oxygen that are
generated in cells under oxidative stress. In addition, MDA is
generated via the autooxidation of AsA (Scarpa et al., 1983)
and via the spontaneous oxidation of AsA by electron carriers such as
cytochrome c, cytochrome b
, and
cytochrome b
. Furthermore, MDA is produced
during the enzymatic reactions catalyzed by AsA oxidase (Yamazaki and
Piette, 1961), thyroid peroxidase (Nakamura and Ohtaki, 1993), and
dopamine
-monooxygenase (Dhariwal et al., 1991).
De-epoxidation of violaxanthine to zeaxanthine in chloroplasts seems to
be associated with a photoprotective function, and MDA is very probably
generated in this reaction since AsA is required for the de-epoxidation
reaction (Yamamoto et al., 1972).
To maintain the antioxidant activity of AsA, the regeneration of AsA from MDA is obviously indispensable. For example, in leaf cells, AsA is found at or above 10 mM in chloroplasts. However, from rates of the photoproduction of superoxide and hydrogen peroxide, it can be calculated that the AsA in chloroplasts is consumed within 80 s if no system in regeneration of AsA is operative in illuminated chloroplasts (Asada, 1994). Furthermore, several enzymes are inactivated by MDA (Davison et al., 1986, Harwood et al., 1986), and the toxicity of AsA to cells (Stich et al., 1976) might be attributable to MDA. Thus, it is essential to maintain a low steady state concentration of the MDA radical in cells.
The NADPH-dependent
activity for regeneration of AsA from MDA has been found in mammalian
tissue, but the enzyme that catalyzes the regeneration has not been yet
purified. Cytochrome b and cytochrome b
reductase can reduce MDA (Iyanagi and Yamazaki,
1969; Nishino and Ito, 1986; Njus and Kelly, 1993), but the reactivity
of MDA with reduced cytochrome b
reductase is
several orders of magnitude lower than that of plant MDA reductase
(Kobayashi et al., 1991). The activity for the reduction of
MDA, with NAD(P)H as the electron donor, has been found in plants, and
it is not only in chloroplasts (Hossain et al., 1984) but also
in nonphotosynthetic tissues (Arrigoni et al., 1981; Bowditch
and Donaldson, 1990) and in algae (Shigeoka et al., 1987;
Miyake et al., 1991). It has also been demonstrated that the
photoreduced ferredoxin in photosystem I of chloroplast thylakoids can
reduce MDA at a high rate (
10
M
s
), which would contribute to the regeneration
of AsA in the thylakoidal system for scavenging of hydrogen peroxide
(Miyake and Asada, 1994).
MDA reductase is the first known enzyme
that uses an organic radical as the substrate, and it catalyzes the
following reaction: 2MDA + NAD(P)H + H
2AsA + NAD(P)
.
The enzyme has been purified from cucumber fruits (Hossain and Asada, 1985), soybean root nodules (Dalton et al., 1992), and potato tubers (Borraccino et al., 1986), and it has been characterized to be an FAD-enzyme with a molecular mass of 47 kDa. We have cloned cDNA for a cytosolic isozyme of MDA reductase from cucumber, and the entire sequence of its amino acid residues has been predicted from the nucleotide sequence. The sequence includes FAD and NAD(P) binding domains but exhibits only limited homology, in terms of amino acid sequence, to other flavoenzymes, even those from plants. It exhibits a greater homology to flavoenzymes from prokaryotes, such as putidaredoxin reductase and rubredoxin reductase (Sano and Asada, 1994). The sequence of amino acid residues of MDA reductase from pea has been deduced from its cDNA, and it also shows limited homology to other flavoenzymes (Murthy and Zilinskas, 1994). Thus, the unusual nature of MDA reductase is emphasized by its substrate, an organic radical, and also by the absence of similar flavoenzymes in mammalian and fungi.
For further characterization of the molecular properties of this enzyme, we established a system for high expression of native MDA reductase of cucumber in Escherichia coli. The coding region of the cDNA for the cytosolic isozyme of MDA reductase was inserted into a plasmid under the control of the T7 promoter, and the MDA reductase produced in E. coli was purified. The present communication describes the high expression of MDA reductase in E. coli, and purification and characterization of molecular properties of the enzyme.
pET-8c
(Studier et al., 1990), in which a unique NcoI site
contains the first codon of the 10 gene adjacent to a T7
promoter, was used as the plasmid vector. pET-8c was digested with BamHI and blunt-ended by treatment with T4 DNA polymerase and
dNTPs. It was digested with NcoI and dephosphorylated with
calf intestinal alkaline phosphatase.
Two DNA fragments described
above had one NcoI cohesive end and one blunt end each and
ligated with T4 DNA ligase. After transformation of competent E.
coli DH5 cells with the ligated DNA, plasmid DNA from
ampicillin-resistant colonies was prepared and examined by restriction
digestion to confirm that the construction was correct. The expression
plasmid is referred to as pET-CMR (Fig. 1).
Figure 1:
Construction of the expression plasmid,
pET-CMR, for the overproduction of MDA reductase of cucumber. A portion
of the cDNA clone with the sequence that encodes the whole protein of
MDA reductase from pCMR31KS was ligated to the plasmid
vector pET-8c via an NcoI site and a blunt end generated at a BamHI site. Solid boxes indicate the DNA fragment
derived from the cDNA of MDA reductase from
cucumber.
The dialyzed enzyme was then applied to a column of Q Sepharose High Performance 26/10 (Pharmacia Biotech Inc.) that had been equilibrated with buffer B. After extensive washing of the column with buffer B, the enzyme was eluted by a 150-ml linear gradient from 0 to 0.2 M KCl in buffer B. The active fractions were pooled, concentrated, and equilibrated with buffer B by ultrafiltration through a PM-10 membrane filter (Amicon). Above column chromatographies were performed with a fast-protein liquid chromatography system (Pharmacia, Uppsala, Sweden).
Figure 2: Detection of MDA reductase of cucumber by Western blotting with antibody against MDA reductase from cucumber fruits after SDS-polyacrylamide gel electrophoresis. The E. coli cells from 1 ml of culture at 37 °C were suspended in 100 µl of loading buffer for electrophoresis and boiled for 3 min. The extract of whole cells (2 µl) was subjected to SDS-polyacrylamide gel electrophoresis. Lanes 1-3, bacteria without plasmids; lanes 4-6, bacteria that harbored pET-CMR; lanes 1 and 4, incubated for 2 h without IPTG; lanes 2 and 5, incubated for 4 h without IPTG; lanes 3 and 5, incubated for 2 h without IPTG and for an additional 2 h in the presence of 1 mM IPTG.
A soluble fraction from the cells transformed with pET-CMR, prepared after incubation for various times in LB medium in the presence and absence of IPTG, catalyzed the NADH-dependent reduction of MDA, but that from cells without the plasmid did not. Although the amount of MDA reductase expressed in E. coli cells in response to IPTG was larger than that in noninduced cells, as determined by Western analysis, higher activity of MDA reductase was found in the soluble fraction when cells were incubated in LB medium for 12 h without the induction by IPTG than after induction by IPTG (data not shown). A larger fraction of the MDA reductase protein, expressed in the induced cells, might have formed insoluble inclusion bodies in the latter case. We observed the maximal activity of MDA reductase under the culture conditions described under ``Materials and Methods.''
A soluble extract of E.
coli cells that harbored pET-CMR and had been cultured in LB
medium at 37 °C for 12 h without induction has a specific activity
of about 40 units mg of protein, which was about
80-fold higher than that in extracts of cucumber fruits. MDA reductase
accounted for nearly 20% of the soluble protein in the E. coli cells. The enzyme was purified to homogeneity by a simple
procedure with a yield of about 45 mg from 2 liters of culture, as
summarized in Table 1. The analysis by SDS-polyacrylamide gel
electrophoresis of the purified enzyme gave a molecular mass of 47 kDa,
as expected from the open reading frame of the cDNA (Sano and Asada,
1994), and the specific activity of the purified enzyme was similar to
that of MDA reductase purified from cucumber fruits (Hossain and Asada,
1985). The purified enzyme was stable for at least 6 months when stored
at -85 °C in 10 mM HEPES-NaOH, pH 7.0, 10 mM 2-mercaptoethanol, and 0.1 mM EDTA. Recombinant MDA
reductase crystallized as yellow needles in a precipitant that
contained 0.2 M calcium acetate, 0.1 M sodium
cacodylate, pH 6.5, and 18% (w/v) polyethylene glycol 8000 over the
course of 2 weeks (Fig. 3).
Figure 3: Crystals of MDA reductase of cucumber highly expressed in E. coli. Bar represents 200 µm.
Figure 4: Anaerobic titration of recombinant MDA reductase with NADH. Oxidized MDA reductase (62.6 nmol) in 1 ml of 50 mM potassium phosphate, pH 7.0, was rendered anaerobic by repeated evacuation and flushing with argon in an optical cell used for anaerobic titration (Iyanagi et al., 1974). Nitrogen was passed at a low rate through the gas lock that protected the cell unit from air, and the enzyme was titrated with 5 mM NADH in the same buffer with a gas-tight Hamilton microsyringe. The absorption spectra from the top to the bottom correspond to those of the enzyme after the addition of 0, 0.16, 0.32, 0.48, 0.64, 0.80, and 0.96 eq of NADH relative to the enzyme.
The fully reduced enzyme was then back-titrated with the MDA radical, which was continuously generated by the AsA-AsA oxidase system (Fig. 5). The reduced enzyme was completely oxidized when 2.18 mol of MDA radicals were generated per mol of MDA reductase. During the oxidation process, the absorption spectrum of the semiquinone form could not be detected, although a blue-shifted peak of the oxidized flavin around 370 nm was observed at an early stage of the oxidation.
Figure 5:
Back-titration of reduced recombinant MDA
reductase with the MDA radical. Oxidized MDA reductase (50.3 nmol) in 1
ml of 50 mM potassium phosphate, pH 7.0, that contained 0.5
mM AsA (spectrum 1), was first reduced with 1.01
molar eq of NADH (spectrum 2). The reduced enzyme was then
titrated with the MDA radical, which was generated by the reaction
catalyzed with 5 microunits of AsA oxidase at 5 nmol min under aerobic conditions, and absorption spectra were recorded 4
min, 8 min, 12 min, 16 min, and 20 min after the addition of AsA
oxidase. Finally, 22 min after the addition of AsA oxidase, when the
accumulated production of MDA had reached 110 nmol, MDA reductase had
the same spectrum as the oxidized enzyme (spectrum 1).
Scanning time: 100 s.
The electron acceptor of MDA reductase is the MDA radical,
and the NADH-reduced enzyme should be oxidized via two successive
oxidations by two MDA radicals. Therefore, the semiquinone form of the
enzyme should be formed as an intermediate, but static titration of the
reduced enzyme did not allow us to show a spectrum of the semiquinone.
Flavoproteins can be reduced by illumination under anaerobic conditions
in the presence of EDTA as the electron donor (Massey and Palmer,
1966). The semiquinone form is stabilized by binding of NAD as a catalytic intermediate, as shown for ferredoxin-NADP
reductase (Keirns and Wang, 1972), adrenodoxin reductase (Kitagawa et al., 1982), and cytochrome b
reductase
(Iyanagi, 1977). The spectrum of the NADH-reduced MDA reductase
indicates the formation of a stable charge-transfer complex (Fig. 4), as in the case of cytochrome b
reductase (Iyanagi, 1977). Therefore, photoreduction of MDA
reductase was performed in the presence of NAD
at a
molar ratio of 1:1 with the enzyme under anaerobic conditions using
EDTA as the electron donor. During illumination, a new spectrum with a
peak at 370 nm and a flat absorption in the long-wavelength region was
generated (Fig. 6). This spectrum is characteristic of
NAD
-bound, red semiquinone forms in flavoenzymes of
the dehydrogenase-oxidase group (Massey and Hemmerich, 1980). Thus, the
NAD
bound semiquinone, which can infer the spectrum
change at an early stage of oxidation of the reduced enzyme by MDA (Fig. 5).
Figure 6:
Photoreduction of recombinant MDA
reductase under anaerobic conditions in the presence of EDTA. MDA
reductase (74.7 nmol) in 1 ml of 50 mM HEPES-NaOH, pH 7.6,
that contained 50 mM EDTA and 75 µM NAD was rendered anaerobic by repeated evacuation
and flushing with argon, and it was illuminated with a 650-watt
tungsten lamp at 10 °C. The absorption spectra of the photoreduced
enzyme were recorded at the indicated times after the start of
illumination.
Preincubation of recombinant MDA reductase with 1.5 mol eq of pCMB for 10 min inhibited the reduction of FAD by NADH, as determined by the decrease in absorbance at 450 nm (Fig. 7), as is the case for the enzyme purified from cucumber fruits (Hossain and Asada, 1985). Thus, one Cys residue seems to participate in the reduction of the enzyme-FAD by NADH. Neither of the two Cys residues in MDA reductase is conserved in other flavin-containing oxidoreductases (Sano and Asada, 1994), and it is not known which Cys residue reacts rapidly with thiol reagents and participates in the transfer of electrons from NADH to the FAD. Cys-198 of the enzyme from cucumber is located near the putative NADH binding domain (Sano and Asada, 1994) and could participate in electron transfer between NADH and the enzyme-FAD.
Figure 7: Blockage of the transfer of electron from NADH to recombinant MDA reductase-FAD by preincubation of the enzyme with pCMB. The spectra of the recombinant MDA reductase (37.3 nmol) were recorded in 1 ml of 50 mM HEPES-NaOH, pH 7.6, under aerobic conditions. Curve 1, oxidized enzyme; curve 2, enzyme that had been fully reduced with 0.99 mol eq of NADH; curve 3, enzyme that had been incubated with 1.5 mol eq of pCMB for 10 min, and then with an additional 0.99 mol eq of NADH.
The K values
for NADH and NADPH of MDA reductases from various plants are summarized
in Table 2. These data allow us to divide MDA reductases into two
groups. The enzyme from soybean root nodules and the recombinant enzyme
from cucumber are characterized by a low specificity for NADPH. Other
MDA reductases, purified from cucumber fruits, spinach, and potato
tubers, gave only severalfold higher values of K
for NADPH than those for NADH. Although the amino acid
sequence of cytosolic MDA reductase predicted from pea cDNA had a high
degree of homology (78%) to that of cucumber, the fusion protein of MDA
reductase of pea with a maltose-binding protein did not show high
specificity for NADH (Murthy and Zilinskas, 1994). The domain of the
maltose-binding protein might affect the interaction of electron donors
with the fused pea enzyme.
Figure 8: Dixon plot of the initial velocity of the reaction catalyzed by recombinant MDA reductase at various concentrations of dicumarol with 2 µM and 3 µM MDA. Assays were carried out under the standard conditions with the addition of dicumarol at indicated concentrations.
The enzyme-bound FAD (E-FAD) is reduced by NADH, and a
charge-transfer complex (E-FADHNAD
) is formed (Fig. 4). The reduced enzyme donates electrons to MDA by two
successive one-electron transfers, and a red semiquinone form (E-FAD
NAD
) is thought to be the
intermediate (Fig. 6). The second order rate constant for the
reduction of the enzyme-bound FAD by NADH (k
) was
determined by a stopped-flow analysis, which was monitored at 452.9 nm
after rapid mixing. When we tried to determine the rate under the
pseudo-first order conditions (10 µM enzyme and 100
µM NADH), almost all of the enzyme-FAD was reduced within
the dead time after the mixing of 450 µs of the instrument. This
result corresponds to a rate above 10
M
s
, and so we
could not determine the rate accurately. The reduction of the
enzyme-FAD by NADH in a second order mode (10 µM enzyme
and 10 µM NADH) allowed us to determine the rate (Fig. 9A). The reciprocal plot of the oxidized E-FAD against time after mixing (Fig. 9B)
gives a straight line, and k
is estimated from its
slope to be 1.25
10
M
s
at pH 7.0. The rate constant was not
affected by buffers, when either 50 mM HEPES-KOH or 50
mM potassium phosphate at pH 7.8 was used. The effect of pH on
the rate of reduction of E-FAD by NADH was determined (Fig. 10). Between pH 5.5 and 7, the rate was highest and
constant, and the rate decreased gradually with increases in pH. It
should be noted that the pK
of cysteine
is around pH 8.5 and either Cys-69 or Cys-198 participates in the
reduction of E-FAD by NADH, as discussed above. It appears,
therefore, that the dissociation of either Cys residue lowers the
interaction of the E-FAD with NADH. The pH optimum of the
overall reaction is in a range from pH 7 to pH 9 (Hossain and Asada,
1985, Dalton et al., 1992), suggesting that the rates of
reactions 2 and 3 are high above pH 7.
Figure 9: Reduction of the FAD of MDA reductase by NADH after rapid mixing. MDA reductase (MDAR) and NADH at equimolar concentrations (10 µM) in 50 mM HEPES-NaOH, pH 7.8, were mixed rapidly in a stopped-flow apparatus, and the reduction of FAD was followed as the increase in transmittance at 452.9 nm. For improvement of the S/N ratio, the figure represents the average of four determinations (A). The reciprocal of the concentration of the oxidized enzyme is plotted against the time after the mixing (B).
Figure 10:
Effects of pH on the reaction rate
constants (k) for the reduction of the FAD of MDA
reductase by NADH. The assay conditions were the same as in Fig. 9, except that the following buffers were used: pH
5.5-6.0, 50 mM MES-KOH; pH 7-8, 50 mM
HEPES-KOH; pH 9, bis-tris propane-KOH.
The maximal rate for
bimolecular collisions is 8.4 10
M
s
in water at 30
°C, and it is independent of their molecular sizes (Marshall,
1978). Assuming that MDA reductase is a sphere and the density of the
enzyme is equal to that of hemoglobin, we can calculate that the ratio
of the area of the isoalloxazine ring of FAD to the total surface of
the enzyme is only 0.03%. Therefore, the maximal collision rate of NADH
with E-FAD is 2.5
10
M
s
. Thus, the
observed value of k
is 44-fold higher than the
estimated collision rate, and the interaction of NADH with E-FAD appears to be facilitated by a mechanism such as the
electrostatic guidance of the electron donor to the FAD. At neutral pH,
NADH is present mostly in an anionic form since its
pK
is 3.9, and ionic interactions are
assumed to participate in the guidance of NADH to E-FAD. To
examine such ionic interactions, k
was determined
at various concentrations of NaCl (Fig. 11). With increases in
ionic strength, the rate of reduction of E-FAD by NADH fell to
8.5
l0
M
s
, with an inflection point at 0.74 (0.5 M NaCl), when the rate was plotted against ionic strength. At
present, it is not known why the plot of k
against
ionic strength shows an inflection at 0.5 M NaCl, but the
conformation change of the enzyme by the salt is likely to affect the
reduction rate by NADH. The rate constant was also lowered in phosphate
buffer at its high concentrations (data not shown). The 1 M
NaCl-suppressed rate was similar to the estimated rate of bimolecular
collisions. The present results support the proposed enhanced
electrostatic guidance of NADH to the isoalloxazine ring of the
enzyme-FAD by positively charged amino acid residues. We have not
identified the participating residues of the enzyme. However, three Lys
residues (159, 161, and 165) and one Arg residue (183) are found around
the putative NADH binding domain of NADH (Lys-162 to Leu-182 and
Met-192 to Asp-195 with a loop between them) of MDA reductase (Sano and
Asada, 1994).
Figure 11:
Effects of NaCl on the reaction rate
constants (k) for the reduction of the FAD of MDA
reductase by NADH at pH 7.8. The assay conditions were the same as in Fig. 9, except for the addition of NaCl at various
concentrations up to 1 M.