L-Ascorbic acid is oxidized by a successive reversible
one-electron transfer process with a free radical intermediate, and
thus the ascorbate redox system consists of L-ascorbic acid, L-ascorbyl free radical, and dehydro-L-ascorbic
acid(1) . In biological systems, the relative steady state
level of L-ascorbic acid and dehydro-L-ascorbic acid
seems to be maintained by ascorbate oxidase(2) ,
glutathione-dependent dehydro-L-ascorbate
oxidoreductase(3) , and NADH-dependent
semidehydro-L-ascorbate oxidoreductase(4) .
The
oxidative degradation of L-ascorbic acid in plants seems to be
controlled by copper-containing ascorbate oxidase(2) . After
the oxidation by ascorbate oxidase, it has been suggested that
dehydro-L-ascorbic acid can be transformed chemically or
through the biological catalytic pathway composed of
2,3-diketo-L-gulonic acid,
-ketoaldehyde, and some
enediol compounds(5, 6) .
Ascorbate oxidase is
found mostly in the peripheral parts of the plant, closely associated
with the cell-wall material during plant growth, such as in the
germination of pumpkin seeds(7, 8) . Although various
biochemical and biological functions of ascorbate oxidase have been
discussed, the in vivo role of ascorbate oxidase in plants is
still under debate.
Ascorbate oxidases, which have been purified
from higher plants, such as green zucchini (2, 9) and
cucumber(10) , catalyze the oxidation of L-ascorbic
acid to dehydro-L-ascorbic acid and belong to blue copper
oxidases including laccase and ceruloplasmin. The refined crystal
structure of the fully oxidized form of ascorbate oxidase from green
zucchini was recently reported(11) .
On the other hand,
there are almost no reports on the oxidation of ascorbic acid in
microorganisms. Even though the occurrence of ascorbate oxidase was
reported in Myrothecium verrucaria(12) and there was
a report on the purification and characterization of the enzyme from
the culture filtrate of Acremonium sp. HI-25, which contains
copper in its active site(13, 14) , the properties of
intracellular ascorbate oxidase in microorganisms are not yet
clarified. In the present study, we report the purification and
characterization of a novel type of an intracellular heme-containing
ascorbate oxidase from an oyster mushroom, Pleurotus
ostreatus.
EXPERIMENTAL PROCEDURES
Materials
L-Ascorbic acid and D-ascorbic acid were purchased from Merck, Sepharose CL-6B,
reactive red 120-Sepharose CL-4B, Sephadex G-200, S Sepharose CL-6B,
and molecular mass markers for high performance gel filtration
chromatography from Sigma, Protein PAK SW300 and Pico-Tag column from
Waters, poly(vinylidene difluoride) membrane from Millipore, and
molecular mass standards for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis from Boehringer Mannheim. L-Erythroascorbic
acid and D-erythroascorbic acid were prepared according to the
methods of Gan and Seib(15) . All other reagents used were of
the highest quality generally available.
Microorganism and Growth Conditions
The mycelium
of white-rot fungus Pleurotus ostreatus, a kind gift from the
Korean Forest Research Laboratory, was grown in complete medium
containing 2% malt extract (w/v), 0.5% peptone (w/v), 0.5% yeast
extract (w/v), and 1% glucose (w/v). It was cultivated for 4 days at 28
°C in a 500-ml Erlenmeyer flask containing 200 ml of medium in a
reciprocally shaking water bath operating at 100 rpm.
Enzyme Assay
The activity of ascorbate oxidase was
determined using 0.1 M sodium phosphate, citrate buffer (pH
5.4) containing 0.5 mM EDTA, according to the method of
Oberbacher and Vines(16) . The oxygen consumption rate was also
measured using Clark-type oxygen electrodes (YSI Instrument Co.). One
unit of enzyme activity was defined as the amount of enzyme required to
catalyze the oxidation of 1 µmol of L-ascorbic acid/min.
For the determination of K
values, the enzyme
activities were assayed at 290 nm, using a molar absorption coefficient
of 2,800 M
cm
(17) , in the concentration range of
0.025-0.75 mM ascorbic acid by means of Shimadzu model
UV-265 spectrophotometer. K
values were determined
from Lineweaver-Burk plots.
Purification of the Enzyme
The mycelia from the
exponential phase were used as a starting material for purification.
All of the purification steps were performed at 4 °C. The grown
mycelia of P. ostreatus were collected by filtration on
Whatman No. 1 filter paper and washed three times with 500 ml of 50
mM potassium phosphate buffer (pH 6.6) containing 0.5 mM EDTA (buffer A). The washed mycelia were homogenized in buffer A
containing aluminum oxide. The aluminum oxide and unbroken mycelia were
removed by centrifugation. The supernatant was used as a crude extract
during the enzyme purification. Cold acetone was added to 65%
saturation to the crude extract and the resulting precipitate was
collected by centrifugation and the residual acetone was purged out
with the nitrogen gas. The precipitate was resuspended in buffer A and
loaded onto a Sepharose CL-6B column (3.3
100 cm) previously
equilibrated with buffer A. The active fractions were concentrated and
changed into 20 mM potassium phosphate buffer (pH 6.6)
containing 0.5 mM EDTA (buffer B) through an Amicon
ultrafiltration system with PM 10 membrane (Amicon). The concentrated
solution was applied on reactive red-Sepharose CL-4B column (2.5
15 cm) equilibrated with buffer B. The active fractions were
concentrated and loaded on a Sephadex G-200 column (2
110 cm)
previously equilibrated with buffer A. The enzyme-containing fractions
were collected and changed into 20 mM sodium phosphate,
citrate buffer (pH 4.3) containing 0.5 mM EDTA (buffer C)
using an Amicon ultrafiltration system with a PM 10 membrane. The
concentrated enzyme was applied onto an S-Sepharose CL-6B column (3
20 cm) previously equilibrated with buffer C. After the column
was washed with buffer C, the bound proteins were eluted with a linear
concentration gradient of 0.15-0.4 M NaCl. The active
fractions were combined by an Amicon ultrafiltration system with a PM
10 membrane, changed into 10 mM sodium phosphate buffer (pH
5.4) without EDTA, and stored at 4 °C.
Determination of Molecular Mass
The molecular mass
of the purified enzyme was determined by high performance gel
permeation chromatography using a Protein PAK SW300 column (7.8
300 mm). The column was calibrated with ovalbumin (43 kDa), bovine
serum albumin (66 kDa), and catalase (240 kDa). For the determination
of the molecular mass of the subunit, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed on slab gels
with a 5-20% linear concentration gradient according to the
method of Laemmli(18) . As standard markers,
-galactosidase (116.4 kDa), fructose-6-phosphate kinase (85.2
kDa), glutamate dehydrogenase (55.4 kDa), lactate dehydrogenase (36.5
kDa), and soybean trypsin inhibitor (20.1 kDa) were used.
Determination of Concentrations of Protein and
Carbohydrate
Protein concentrations were determined according to
the methods proposed by Bradford (19) and Lowry et
al.(20) , using bovine serum albumin as a standard
protein. The carbohydrate content was determined according to the
method of Dubois et al.(21) , using glucose as a
standard carbohydrate.
Amino Acid Analysis
The amino acid analysis was
performed by reverse-phase chromatography after phenylisothiocyanate
derivatization according to the method proposed by Bidlingmeyer et
al.(22) . The derivatized amino acids were analyzed using
a reverse-phase high performance Pico-Tag column. After sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of the enzyme,
electrotransfer of protein was carried out according to the method of
Towbin et al.(23) . The N-terminal amino acid sequence
was determined by means of Milligen/Biosearch 6600 Prosequencer
protein-sequencing system from Millipore.
Spectroscopic Studies
UV absorption spectra were
obtained in 0.1 M phosphate buffer (pH 6.0) at 25 °C with
a Shimadzu model UV-265 spectrophotometer. Reduced-minus-oxidized
difference spectra were obtained by recording the spectrum of the
enzyme reduced with 5 mM ascorbic acid and a few crystals of
solid Na
S
O
, and the spectrum of the
sample was oxidized by bubbling oxygen for 20 min. For ligand-binding
study, the samples were reduced with a few granules of
Na
S
O
, making an anaerobic condition
by exchanging the sample headspace with argon and then gently mixing.
After several cycles of argon exchange, CO was passed through the
sample solution and (reduced-plus-CO)-minus-reduced difference spectra
were obtained. The spectra of potassium cyanide complex were recorded
through the same procedure. The spectra of pyridine hemochrome were
obtained according to the method proposed by Berry and Trumpower (24) , and the content of protoheme was determined at 562 nm
using a molar absorption coefficient of 30,000 M
cm
(25) .
Stoichiometric Determination
All experiments for
stoichiometric analysis were performed at 25 °C using 0.1 M phosphate citrate buffer (pH 5.4) containing 0.5 mM EDTA.
The reaction was monitored using an UV-visible spectrophotometer and
oxygen monitor. The concentration of hydrogen peroxide was determined
according to the method of Nishikimi et al.(26) .
Analysis of Reaction Product
To 3 ml of the enzyme
solution (0.25 mg/ml), 3 mg of L-ascorbic acid were added, and
this mixture was flushed with oxygen at 37 °C for 1 h. The reaction
process was traced by measuring the absorbance at 265 nm every 10 min
during the reaction. After the oxidation, the solution was freeze-dried
immediately. The freeze-dried preparation was redissolved in 0.5 ml of
D
O.
H NMR spectra of enzymatic reaction
products dissolved in D
O were obtained at room temperature
by means of VXR-200S FT-NMR spectrometer from Varian, using
3-(trimethylsilyl)-1-propane sulfonic acid as an internal reference.
RESULTS AND DISCUSSION
Purification of the Enzyme
The
ascorbate-oxidizing activity in aerobically grown P. ostreatus was detectable in the mycelium from the lag phase to the
exponential phase. The activity of ascorbate oxidase was overlapped
with that of laccase, because laccase occurs in the mycelial crude
extract of P. ostreatus and can oxidize ascorbic acid.
However, after the first gel filtration was performed, the enzyme
activity was reproducible. Ascorbate oxidase was purified from P.
ostreatus, as summarized in Table 1. The enzyme was purified
420-fold relative to the crude cell extract with a recovery of 13%. The
purity of the enzyme was confirmed by an electrophoretic method. The
preparation of the purified enzyme gave a single band with enzyme
activity after nondenaturing polyacrylamide gel electrophoresis.
Molecular Properties
The apparent molecular mass
of the purified enzyme was determined to be 94 kDa by high performance
gel permeation chromatography. On the other hand, when the enzyme was
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
a single band of 46 kDa was found (Fig. 1), indicating that the
enzyme is composed of two identical subunits. The ascorbate oxidases
from cucumber and squash are dimeric enzymes with a molecular mass in
the range of 66-70 kDa for the monomer, while the enzyme from the
culture filtrate of Acremonium was a monomer with a molecular
mass of 80 kDa(13) .
Figure 1:
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of ascorbate oxidase. Lane 1, purified
enzyme; lane 2, molecular mass markers;
-galactosidase (a, 116.4 kDa), fructose-6-phosphate kinase (b, 85.2
kDa), glutamate dehydrogenase (c, 55.4 kDa), lactate
dehydrogenase (d, 36.5 kDa), and soybean trypsin inhibitor (e, 20.1 kDa). Relative mobilities of the standard markers versus common logarithms of their molecular weights were
plotted. With the linear regression output, the molecular weight of
ascorbate oxidase was estimated.
The pI value of this enzyme is 4.5. The
ascorbate oxidase contains approximately 12.5% carbohydrate by weight,
which is estimated according to the method proposed by Dubois et
al.(21) . All of the ascorbate oxidases previously
reported were also glycoproteins: ascorbate oxidase from zucchini
contains 10% carbohydrates (27) and has two N-linked
oligosaccharide chains per subunit (28) .
Amino Acid Composition and N-terminal Amino Acid Sequence
of the Enzyme
As shown in Table 2, the enzyme contained
very different compositions of amino acids from those of ascorbate
oxidases from green zucchini (9) and Cucumis
sativus(29) . And the N-terminal amino acid sequence of
the enzyme is
Asp-Val-Lys-Thr-Leu-Gln-Glu-His-Leu-Gln-Leu-Ala-Leu-Met-Val-. As shown
in Fig. 2, this sequence is also very different from those of
ascorbate oxidases from Cucurbita pepo medullosa(11) , Cucumis sativus(29) , and Cucurbita sp. Ebisu Nankin(30) .
Figure 2:
The comparison of N-terminal amino acid
sequences between several ascorbate oxidases. a, data from
Ohkawa et al.(29) ; b, data from
Messerschmidt et al.(11) ; and c, data from
Esaka et al.(30) .
Effects of pH and Temperature
The enzyme
exihibited maximum activity at pH 4.8-5.5 (Fig. 3A). L-Ascorbic acid can be easily
autooxidized at alkaline pH, thus the enzymatic oxidation can be
distinctly observed only at acidic pH. The enzyme was relatively stable
at acidic pH. The highest rates of the enzymatic reaction were observed
at 40 °C, and above 60 °C the enzyme lost its activity. When
the enzyme was incubated at various temperatures for 1 h at pH 5.4, it
was found to be stable below 50 °C, and 95% of the initial activity
was lost at 55 and 60 °C, respectively (Fig. 3, B and C).
Figure 3:
Effect of pH and temperature on the
activity and stability of ascorbate oxidase. A, changes of the
enzyme activity measured in each pH buffer solution at 37 °C (solid line) and those measured in 0.1 M phosphate
buffer (pH 5.4) at the same temperature, after the incubation in each
pH buffer solution for 12 h (dashed line). B, changes
of the enzyme activity measured at each temperature using 0.1 M phosphate citrate buffer (pH 5.4) and C, those assayed
with the same buffer at 37 °C, after the incubation at each
temperature for 1 h.
Kinetic Calculations
The relationship between
enzyme activity and substrate concentration was Michaelis-Menten type.
The K
and V
values for L-ascorbic acid determined from Lineweaver-Burk plot were
estimated to be 0.48 mM and 1.4 µM min
, respectively. The oxidation rate of L-ascorbic acid by the purified enzyme was compared with
various cytochromes to confirm whether the enzyme was another kind of
cytochrome. The purified enzyme exhibited much higher affinity toward
ascorbate than any other cytochromes (Table 3). Ascorbate oxidase
from P. ostreatus has similar kinetic parameters relative to
those observed with purified enzyme from Acremonium sp. K
, and k
values of the
former for L-ascorbic acid were 0.48 mM and 1.1
10
s
, respectively (Table 4, B), and those of the latter 0.29 mM and 3.1
10
s
, respectively(14) .
Substrate Specificity
As shown in Table 4,
the ascorbate analogs such as L-erythroascorbic acid, D-ascorbic acid, and D-erythroascorbic acid were
oxidized, but the substances which have affinity for laccase did not
serve as substrates. While the ascorbate oxidase from plant can
catalyze various substances, such as reductic acid, catechol, or
dichlorohydroquinone(31) , the ascorbate oxidase from P.
ostreatus is more specific for L-ascorbic acid and its
analogs. D-Ascorbic acid differs only in the configuration of
C-5 from L-ascorbic acid and D-erythroascorbic acid
is a five-carbon analog of L-ascorbic acid, and its C-4
configuration is the same as L-ascorbic acid. L-Erythroascorbic acid is a 4-epimer of D-erythroascorbic acid. D-Ascorbic acid and D-erythroascorbic acid which share similar conformation to L-ascorbic acid in the C-4 position have K
values in the 0.4-0.5 mM range, whereas the
somewhat different L-erythroascorbic acid has a K
of 2.45 mM. These data indicate that
the C-4 configuration of the substrate is essential for its binding to
the enzyme.
Effects of Various Compounds and Metal Ions
The
effects of the various compounds listed in Table 5on the enzyme
activity were examined using L-ascorbic acid as a substrate.
1,10-Phenanthroline caused complete inhibition at the concentrations
shown in Table 5. The straight lines obtained in the double
reciprocal plot, 1/V against 1/[ascorbate] at
different 1,10-phenanthroline concentrations, crossed at a point (Fig. 4A), whereas linear lines were obtained for a
plot of 1/V against [1,10-phenanthroline] (Fig. 4B). This reaction mode accounts for the
competitive inhibition, and from these results, K
was calculated to be 8.3 µM. Sulfhydryl reagents,
such as iodoacetate and p-chloromercuribenzoate, showed no
inhibition. This result supports the conclusion that ascorbate oxidase
from plants is not sulfhydryl-dependent. Also, the enzyme was inhibited
by azide and cyanide.
Figure 4:
Inhibition of ascorbate oxidase by
1,10-phenanthroline. A, Lineweaver-Burk plot for ascorbic acid
as a function of the concentration of 1,10-phenanthroline, and B, slopes from A are replotted against concentrations
of 1,10-phenanthroline.
Spectroscopic Studies
The final homogeneous
preparation of the enzyme was brownish red in solution, suggesting the
presence of the heme group in its active site. The absorption spectra
of the purified enzyme revealed a Soret maximum at 418 nm in its
oxidized form, at 426 nm in its reduced form, and
and
bands
at 558 and 527 nm only in its reduced form, respectively, as shown in Fig. 5. Judged from the position of
band in the difference
spectrum of the reduced-minus-oxidized enzyme, this enzyme seems to
belong to the group of b-type cytochromes, and the absorption
maximum at 562 nm observed in the pyridine hemochrome spectrum is
indicative of a protoheme (data not shown). This spectrum was obtained
through the acid/acetone extract from an ether-soluble heme fraction.
The solubility in ether confirmed that the enzyme belongs to the group
of b-type cytochromes, not covalently bound to the enzyme. On
the basis of the molar absorption coefficient for the
band of the
reduced-minus-oxidized pyridine hemochromogen, a quantity of 22.5 nmol
of protoheme per mg of protein was calculated. Given the molecular mass
of 94 kDa, 2.12 mol of protoheme per enzyme were present. Therefore,
assuming that a protoheme is not removed during purification
procedures, the ascorbate oxidase from P. ostreatus may
contain one protoheme per monomeric subunit. Calculation of the molar
absorption coefficient of the ascorbate oxidase at 562 nm, by using the
heme concentration derived from the pyridine hemochromogen spectra,
gave a value of 30,000 M
cm
. Absorption spectra of CN-enzyme were
recorded after the addition of 30 mM sodium cyanide to the
oxidized ascorbate oxidase (Fig. 6). The ascorbate oxidase from P. ostreatus was inhibited by cyanide as in cases of
conventional hemoproteins, and the characteristic spectrum of the
cyanide complex was observed, indicating that the heme is the binding
site of cyanide. (Reduced plus CO)-minus-reduced spectra were obtained
by bubbling with a steady stream of CO (Fig. 7). It is somewhat
strange that the Soret band did not shift but increased. This result
corresponds to a previously reported spectrum(32) , but
provides no clue to the nature of CO-heme adduct. Additionally, any
evidence of inhibition by CO was not observed.
Figure 5:
Absorption spectra of ascorbate oxidase. A, spectra of oxidized (solid line) and reduced form (dashed line) of the enzyme. B, difference spectrum
of reduced minus oxidized enzyme. Purified enzyme (2 µM)
was dissolved in 0.1 M sodium phosphate buffer (pH 6.0) and
reduced by 5 mML-ascorbic
acid.
Figure 6:
Absorption spectra of CN-treated ascorbate
oxidase. CN-enzyme (dashed line) was prepared by the addition
of 30 mM sodium azide to the 1.48 µM native
enzyme solution (solid line) in 0.1 M sodium
phosphate buffer, pH 6.0. The right inset shows the 5-fold
enlarged spectra in the 500-600 nm
region.
Figure 7:
CO
difference spectra of ascorbate oxidase. A, CO-binding spectra
were obtained by sodium dithionite in an anaerobic condition. Time
interval was 2 min in the spectra above. B, difference
spectrum of (reduced plus CO)-minus reduced
enzyme.
Stoichiometry
The ascorbate oxidase from P.
ostreatus was estimated to be capable of using only molecular
oxygen, not cytochrome and ferricyanide as an electron acceptor. The
reaction catalyzed by the ascorbate oxidase was L-ascorbic
acid + O
dehydro-L-ascorbic acid
+ H
O
, which is confirmed by the result
shown in Table 6. The purified enzyme was rather inhibited by
hydrogen peroxide. In this study, it has been shown conclusively that
the enzyme really does use molecular oxygen as an electron acceptor,
and oxygen is converted to hydrogen peroxide (Fig. 8).
Figure 8:
Monitoring of oxygen consumption during
the enzymatic oxidation of L-ascorbic acid. At the left-hand arrow, 2.5 µg of ascorbate oxidase were added to
0.6 ml of reaction solution containing 5 mM ascorbic acid in
0.1 M phosphate citrate buffer (pH 5.4), and 2.5 units of
catalase were added at the right-hand arrow. Oxygen
consumption was measured at 25 °C using a Clark-type oxygen
electrode.
Analysis of the Reaction Product
In the
H NMR spectrum of the reaction mixture of L-ascorbic acid with ascorbate oxidase in D
O, the
H-4 resonance peak was obscured by a strong solvent peak. The strong
geminal coupling between H-6 protons indicates that the hydroxymethyl
group at C-6 does not freely rotate. From these results, it is
concluded that dehydro-L-ascorbic acid exists in the bicyclic
hydrated monomer through hemiketal linkage between 6-OH and C-3, as
reported previously(1) .
Conclusion
Considering that ascorbate oxidase is
detected only in the mycelial stage of P. ostreatus and
produces hydrogen peroxide as a reaction product, this enzyme might
play an important role in catalyzing oxidation reactions during
development of this mushroom.Ascorbic acid is well known as a
scavenger for active oxygen species, such as superoxide, hydroxyl
radical, and hydrogen peroxide, especially in the chloroplast and in
the nitrogen-fixing root nodules. That function, however, is mediated
by another enzyme, ascorbate peroxidase (33) . Ascorbate
oxidase is unlikely to be involved in scavenging activity because the
reaction consumes molecular oxygen, rather than hydrogen peroxide.
Considering that water is one of the reaction products of
copper-containing ascorbate oxidase, it is noteworthy that the
heme-containing ascorbate oxidase from P. ostreatus produces
hydrogen peroxide. The electron transfer mechanism in the reaction of
the ascorbate oxidase from P. ostreatus might be different
from that of the copper-containing one.