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INTRODUCTION |
Cellulose, which consists of linear polymers of 1,4-linked
-D-glucose units, is the predominant structural
component of plant cell walls and is estimated to account for about
half of the organic material in the biosphere. Because of this
abundance, cellulose biodegradation has a great impact on the global
carbon cycle and recovery of natural resources. Cellulose
biodegradation by filamentous fungi has generally been considered to
involve only three types of hydrolytic enzymes, i.e.
endo-1,4-
-D-glucanases, 1,4-
-D-glucan cellobiohydrolases, and 1,4-
-D-glucosidase (1). Eriksson
et al., however, demonstrated that the degradation rate of
cellulose by a cell-free culture of the white-rot fungus
Sporotrichum pulverulentum (Phanerochaete
chrysosporium) was higher in the presence of oxygen than in its
absence, suggesting the participation of some oxidation-reduction reaction (2).
In the course of cellulose degradation, many cellulolytic fungi produce
extracellular cellobiose-oxidizing enzymes as well as
cellulose-hydrolyzing enzymes such as cellulases (3-5). Cellobiose dehydrogenase (CDH,1 EC
1.1.99.18) is a flavohemoprotein that oxidizes cellobiose using
molecular oxygen as an electron acceptor (3). It was formerly known as
cellobiose oxidase (CBO, EC 1.1.3.25), but Fe(III)-containing compounds
and quinones were found to have much higher affinity for this enzyme
than does molecular oxygen (6, 7) so CBO was recently renamed CDH (8).
Recent investigations have also demonstrated that another
cellobiose-oxidizing flavoprotein, cellobiose:quinone oxidoreductase
(CBQ, EC 1.1.5.1) (9, 10), is the flavin-containing domain of CDH
produced by proteolytic activity (11-13). In addition to their
catalytic function, both CDH and CBQ can bind to cellulose as well as
many cellulases (11, 14). It was reported that the cellulose-binding
site is located on the flavin domain but not in the catalytic site,
because the enzyme bound to cellulose can still oxidize cellobiose
(14). Moreover, CDH adsorption on cellulose is also observed during cellulose degradation in vivo, especially at cracks on the
cellulose surface formed by cellulases (15). Although the physiological function of this enzyme has not yet been clarified, there is no doubt
that CDH contributes to cellulose biodegradation.
CDH oxidizes the reducing-end groups of cellobiose, higher
cellodextrins, and even cellulose to the corresponding
-lactones in
the presence of electron acceptors (3, 16). Considering the
cellobiose-oxidizing ability of both CDH and its flavin domain (CBQ),
the flavin cofactor appears to be directly responsible for the
oxidation of cellobiose (11). Concomitantly with cellobiose oxidation,
CDH can reduce both Fe(III)-containing compounds and quinones
effectively; however, the reduction processes are significantly different. The reduction rate of cytochrome c by CDH was
reported to be extremely higher than that by the flavin domain, whereas the rates of DCPIP reduction by CDH and the flavin domain were similar
(17). It has been reported that both cytochrome c and DCPIP
were reduced by CDH at pH 4.2, whereas only DCPIP was reduced effectively at pH 5.9. This phenomenon was explained by stopped-flow kinetic study that both flavin and heme were reduced at a high rate at
pH 4.2, whereas only flavin reduction proceeded quickly at pH 5.9 (18).
From these observations, the reduction of cytochrome c is
dependent on heme, and an electron is transferred from cellobiose to
this electron acceptor via both FAD and heme, whereas the reduction of
DCPIP is catalyzed only by flavin.
The thermophilic soft-rot fungus Humicola insolens also
produces CDH under cellulolytic conditions, as do other cellulolytic fungi (19). However, this fungus produces CDH that has a neutral pH
optimum in cytochrome c reduction, whereas CDH produced by other fungi have the optimum at acidic pH and show no activity at
around neutral pH. In this study, therefore, CDH was purified from
P. chrysosporium and from H. insolens, and the
differences between two enzymes were investigated.
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EXPERIMENTAL PROCEDURES |
Materials
P. chrysosporium CDH was purified from the medium of
a cellulose-grown culture of P. chrysosporium as described
by Samejima et al. (20). H. insolens CDH was
purified from CelluzymeTM CAX63 (Novo Nordisk A/S, Bagsvaerd, Denmark)
by using the same procedure. The purity of P. chrysosporium
and H. insolens CDH preparations was confirmed by SDS-PAGE,
isoelectric focusing, and the Rz value (A421/A280).
D-Cellobiose was purchased from ICN.
2,3-Dimethoxy-5-methyl-1,4-benzoquinone (ubiquinone) and bovine heart
cytochrome c were purchased from Tokyo Chemical Industries
Co., Ltd. (Tokyo, Japan). Dithiothreitol was purchased from Wako Pure
Chemical Industries. Ltd. (Osaka, Japan). Papain was purchased from
Elastin Products Co., Inc. (MO). FAD was purchased from Sigma.
6-Hydroxy-FAD was a generous gift from Dr. Vincent Massey, the
University of Michigan. Calcium chloride (suprapure grade) was
purchased from Merck (Darmstadt, Germany).
Methods
Enzyme Assays--
Enzyme activities were assayed as described
by Samejima and Eriksson (7). Ubiquinone (500 µM)
reduction was monitored photometrically by following the absorbance at
406 nm (
= 0.745 mM
1 cm
1).
Cytochrome c reduction was assayed by monitoring the
increase of absorbance at 550 nm (
= 22.4 mM
1 cm
1). Spectrophotometric
experiments were performed with a Shimadzu UV-1600PC spectrophotometer.
The following buffers (50 mM solutions) were used for the
pH dependence studies of enzyme activity and midpoint potential of the
heme domain: sodium citrate (pH 3.0-4.0), sodium acetate (pH
4.0-6.0), MES (pH 6.0, 6.5), HEPES (pH 7.0, 7.5), EPPS (pH 8.0, 8.5),
and CHES (pH 9.0).
SDS-PAGE and Isoelectric Focusing--
SDS-PAGE was carried out
as described by Laemmli (21) using 10% polyacrylamide gel and a
Mini-PROTEAN® II Cell (Bio-Rad). Ultrathin-layer
isoelectric focusing was done essentially as described by Radola (22)
using 5% polyacrylamide gel with 2% Ampholite and a Model 111 Mini
IEF Cell (Bio-Rad).
Limited Proteolysis and Isolation of the Flavin and Heme
Domains--
Limited proteolysis of CDH by papain was performed
essentially as described by Henriksson et al. (11). CDH (20 mg) was incubated in 25 ml of 20 mM phosphate buffer, pH
7.0, containing 2 mM dithiothreitol and 0.5 mg of papain
for 6 h, and then the solution was applied to a DEAE-Toyopearl
650S (Tosoh Co. Ltd., Tokyo, Japan) column (11 × 120 mm)
equilibrated with 20 mM phosphate buffer, pH 7.0, and
eluted with a 150-ml linear gradient of KCl (0-250 mM) in the same buffer. The fractions containing the heme domain were concentrated by ultrafiltration (Ultrafree CL, Millipore Co.) and then
used for experiments. The fractions containing the flavin domain, which
had cellobiose-ubiquinone activity, were pooled and further purified by
hydrophobic chromatography. The buffer solution was changed to 20 mM phosphate buffer containing 1 M ammonium
sulfate, pH 7.0, and then the flavin domain sample was applied to a
column of phenyl-Toyopearl 650M (16 × 120 mm) equilibrated with
the same buffer and eluted with a 500-ml reverse gradient to 20 mM phosphate buffer. The ubiquinone-active fractions were pooled and concentrated by ultrafiltration as described above.
Measurement of Midpoint Potential of the Heme Domain--
A
direct electrochemical technique was used to measure the pH dependence
of the oxidation reduction potential of the heme domain as described by
Hagen (23). Glassy carbon, platinum, and Ag/AgCl were used as the
working, counter, and reference electrodes, respectively. Cyclic
voltammetry was performed in the presence of 50 mM
MgCl2. The midpoint potential was determined by averaging the anodic and cathodic peak potentials.
Identification of the Prosthetic Group of the Flavin
Domain--
Flavin extraction from the flavin domain was based on the
cold trichloroacetic acid method. That is, 100 µl of 50 µM flavin domain, cooled on ice, was mixed with an equal
volume of ice-cold 20% trichloroacetic acid aqueous solution. The
mixture was kept on ice for 10 min and centrifuged to remove the
precipitate. The supernatant was neutralized by the addition of 1300 µl of 1 M phosphate buffer, pH 7.0. The samples were
applied to a Sep-Pak C-18 cartridge (Millipore Co.), eluted with 50%
methanol, 10 mM sodium acetate buffer, pH 5.0, and
concentrated by evaporation using a nitrogen gas stream. Flavin was
analyzed by high-performance liquid chromatography with a JASCO-HPLC
system (Jasco Co. Ltd., Tokyo, Japan) equipped with a Supelcosil
LC-18-T column (4.6 × 250 mm; Supelco, Inc., PA) with a linear
gradient of 20-30% methanol, 10 mM sodium acetate buffer,
pH 5.0, and flavin was detected by measuring the absorption at 440 nm,
which is the isosbestic point of the anionic and protonated forms of
6-hydroxy-FAD. Authentic FAD and 6-hydroxy-FAD were used as standards
for identification and quantitation of flavin under the same conditions.
Preparation of Deflavo-CDHs and Reconstitution with
Flavins--
Preparation of deflavo-CDH was based on the method of
Komai et al. (24). That is, 500 µl of 10 µM
CDH, containing 5 mM cellobiose, 5 mM
dithiothreitol and 3.0 M (P. chrysosporium) or
2.5 M (H. insolens) CaCl2 in 50 mM sodium acetate buffer, pH 4.0 (P. chrysosporium), or HEPES buffer, pH 7.5 (H. insolens),
was kept on ice for 60 min, and then the solution was passed through a
desalting column (Ampure SA; Tosoh Co. Ltd., Tokyo, Japan) equilibrated
with 50 mM HEPES buffer, pH 7.5, to stop the reaction and
to remove low molecular weight materials. The obtained deflavo-CDH (1 µM) was kept on ice. For reconstitution, deflavo-CDH was
incubated with 2 µM flavin, FAD, or 6-hydroxy-FAD, and 5 mM dithiothreitol in 50 mM sodium acetate
buffer, pH 4.0 (P. chrysosporium) or HEPES buffer, pH 7.5 (H. insolens) on ice for 60 min. The reconstituted preparations were concentrated by ultrafiltration as described above to
remove excess free flavin and dissolved in the same buffers. The flavin
domain was prepared by limited proteolysis as described in a previous
section. For spectrophotometric determination, the deflavo-flavin
domain was prepared by the same procedure but without flavin
reconstitution. Native, deflavo-, and reconstituted flavin domains
showed the same molecular weight on SDS-PAGE, indicating that the
treatments had no effect on the proteolytic site.
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RESULTS |
Purification, Characterization, and Limited Proteolysis of
CDH--
CDH purified from culture solution of P. chrysosporium gave a single band at 90 kDa on SDS-PAGE (Fig.
1, lane 1), the isoelectric point was 4.2, and the Rz value
(A421/A280) was 0.62. During purification, it was found that crude cellulase powder from
H. insolens contained three CDH fractions: one major
fraction (94 kDa, pI 4.4, and Rz 0.63) and two
minor fractions (both 92 kDa, pI 4.0, and Rz
0.63). Although all CDH was eluted in the same fraction in the first strong anion-exchanging chromatography (QAE-Toyopearl 550C), it was
separated into two fractions, 94- and 92-kDa fractions by weak
anion-exchanging chromatography (DEAE-Toyopearl 650S) as reported by
Schou et al. (19). However, the 92-kDa fraction was further
separated into two minor CDHs by hydrophobic chromatography (phenyl-Toyopearl 650M). In this study, the major fraction of CDH (94 kDa) was used for further investigation as CDH from H. insolens.

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Fig. 1.
SDS-PAGE of purified CDHs and their
proteolytic fragments. S, molecular weight standards;
lane 1, P. chrysosporium CDH; lane 2,
flavin domain of P. chrysosporium CDH; lane 3,
heme domain of P. chrysosporium CDH; lane 4,
major fraction of H. insolens CDH; lane 5, flavin
domain of H. insolens CDH; and lane 6, heme
domain of H. insolens CDH. Aliquots of 2 µg of each sample
prepared by the methods as described under "Experimental
Procedures" were loaded on 10% polyacrylamide gel.
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The CDHs from P. chrysosporium and H. insolens
had different values of pH optimum when the pH dependence of cytochrome
c reducing activity was monitored using cellobiose as a
substrate (Fig. 2); CDH from H. insolens had the pH optimum around pH 7.5-8.0, whereas CDH from
P. chrysosporium had the optimum at pH 3.5-4.0. The
specific activity of H. insolens CDH at pH 7.5 was 3.3 s
1 and was 10-fold lower than that of P. chrysosporium CDH at pH 4.0 (37 s
1). Absorption
spectra of the oxidized CDHs are shown in Fig.
3. Both enzymes showed typical
hemoprotein spectra having an absorption maximum at 421 nm (
band).
It should be noted, however, that minor differences in the region of
450-500 and 300-400 nm were found between the two CDHs because of the
difference in the flavin chromophore, as described below. The spectral
patterns of the two minor fractions of CDH from H. insolens
described above were similar to that of the major fraction (data not
shown), suggesting that all CDHs from H. insolens have the
same flavin chromophore.

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Fig. 2.
pH dependence of cytochrome c
reducing activity of P. chrysosporium and
H. insolens CDH with cellobiose as the substrate.
, P. chrysosporium; , H. insolens. Activity
was monitored by measuring the increase of absorbance at 550 nm at
various pH values in the buffer systems described under "Experimental
Procedures."
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Fig. 3.
Absorption spectra of P. chrysosporium
and H. insolens CDH. Dotted
line, P. chrysosporium; solid line, H. insolens. Spectra of the purified enzymes were obtained in 20 mM HEPES buffer, pH 7.5.
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To clarify the reason for the spectral difference, limited proteolysis
of the two CDHs with papain was carried out to isolate the heme and
flavin domains for further investigation. As shown in Fig. 1, two bands
were isolated and purified from each of the CDHs. From the absorption
spectra of each band, as described in the following sections, it was
concluded that the bands corresponding to molecular masses 35 and 55 kDa were the heme and flavin domains of P. chrysosporium
CDH, respectively, in accordance with the results reported by
Henriksson (17). On the other hand, the bands corresponding to
molecular masses 25 and 70 kDa were the heme- and flavin-containing
domains from H. insolens CDH, respectively, indicating that
proteolysis occurred similarly between the heme and flavin domains but
the cleavage site was somewhat different from that of P. chrysosporium CDH.
Absorption Spectrum and Midpoint Potential Comparison of Heme
Domains--
The absorption spectra of the oxidized heme domains
separated from P. chrysosporium and H. insolens
CDH are shown in Fig. 4. No particular
difference was observed in the oxidized heme spectra of the two CDHs.
Moreover, no difference in the reduced spectra obtained by addition of
sodium dithionite was observed either (data not shown). No significant
difference in the redox properties of the heme domains was revealed by
determination of the pH dependence of the midpoint potential of each
heme domain using a direct electrochemical technique, as shown in Fig.
5. The potentials of the P. chrysosporium and H. insolens heme domains were 190 and
185 mV at pH 3.0, respectively, then declined until around pH 4.0-5.0,
and became pH-independent over pH 6.0. The potentials of the P. chrysosporium and H. insolens heme domains at pH 7.0 were 130 and 126 mV, respectively. The value for the P. chrysosporium enzyme at pH 4.0 is 180 mV, which is in good agreement with the previously published value of 164 mV obtained by a
dye-mediated optical redox titration (6).

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Fig. 4.
Absorption spectra of the purified heme
domains of P. chrysosporium and H. insolens
CDHs. Dotted line, P. chrysosporium;
solid line, H. insolens. Spectra of the same
samples as described in Fig. 1 were recorded in 20 mM HEPES
buffer, pH 7.5.
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Fig. 5.
pH dependence of midpoint potential of the
heme domains measured by a direct electrochemical technique. ,
P. chrysosporium; , H. insolens. Cyclic
voltammograms of 1.0 mg/ml heme domains were taken from +300 to 300
mV versus Ag/AgCl at various pH values in the buffer systems
described under "Experimental Procedures."
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Characterization and Comparison of the Flavin Domains--
The pH
dependence of the cellobiose-oxidizing activity of CDH and the flavin
domain was monitored in terms of the reduction rate of ubiquinone (Fig.
6). As in the case of P. chrysosporium, the flavin domain of H. insolens CDH had
cellobiose-oxidizing activity at almost the same level as that of
intact CDH, whereas the heme domains of P. chrysosporium and
H. insolens CDH had no activity. Thus, it is concluded that
the catalytic site of cellobiose oxidation is in the flavin domain of
each CDH, and the difference in pH dependence of CDH activity is solely
because of the flavin domains.

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Fig. 6.
pH dependence of cellobiose oxidation by CDHs
and flavin domains from P. chrysosporium
(A) and H. insolens
(B). , CDH; , flavin domain.
Cellobiose oxidation was monitored in terms of the reduction of
ubiquinone by measuring the decrease of absorbance at 406 nm at various
pH values in the buffer systems described under "Experimental
Procedures."
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In contrast to the heme domains, the absorption spectra of the flavin
domains were considerably different between the two CDHs (Fig.
7). The flavin domain from P. chrysosporium showed yellow coloration and had absorption maxima
at 387 and 457 nm, typical of flavin (Fig. 7, inset). On the
other hand, the flavin domain from H. insolens, showed a
green coloration with absorption maxima at 343 and 426, resembling the
spectrum of 6-hydroxyflavin, but a broad peak was observed at around
660 nm at pH 7.5. The long-wavelength absorption peak disappeared at pH
5.0, and the color changed to yellow, consisting with 6-hydroxyflavin
derivatives. The long-wavelength absorption was shifted to 660 nm in
the flavin domain of H. insolens CDH (Fig. 7), as compared
with the broad peak at 600 nm of free 6-hydroxy-FAD (Fig. 7,
inset).

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Fig. 7.
Absorption spectra of flavin domains from
P. chrysosporium and H. insolens.
Dotted line, P. chrysosporium; solid
line, H. insolens. For comparison, the spectra of
authentic FAD (dotted line) and 6-hydroxy-FAD (solid
line) are shown in inset. Spectral measurement was
carried out in 20 mM HEPES buffer, pH 7.5.
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Identification of the Prosthetic Group of the Flavin
Domain--
The prosthetic group of the flavin domain, extracted by
treatment with trichloroacetic acid, was analyzed by HPLC (Fig.
8). The isolated flavin was
non-fluorescent even after phosphodiesterase treatment. A comparison of
the retention time with those of authentic FAD (7.5 min) and
6-hydroxy-FAD (14.0 min), indicated that the flavin domain from
P. chrysosporium contained at least 95% FAD, whereas the
H. insolens flavin domain contained a mixture of 60% 6-hydroxy-FAD and 40% FAD. After treatment of authentic FAD and 6-hydroxy-FAD with phosphodiesterase, the samples were subjected to
HPLC. The retention times of the phosphodiesterase-treated authentic
FAD and 6-hydroxy-FAD were shifted from and to 8.5 and 15.5 min,
respectively, because of the conversion from FAD- to FMN-type
compounds. When the flavin cofactors from CDHs were also treated with
phosphodiesterase, their retention times were identical with those of
the authentic FMNs. Thus, it was concluded that the flavin cofactors
from CDHs were FAD forms and that 6-hydroxy-FAD was the dominant
cofactor of H. insolens CDH.

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Fig. 8.
HPLC analysis for the identification and
quantification of flavin extractives from the flavin domains of
P. chrysosporium and H. insolens. As a standard, 25 µl of a mixed sample of
authentic FAD and 6-hydroxy-FAD (each 50 µM, final) was
applied to HPLC. For detection, the absorbance at 440 nm was followed
as described under "Experimental Procedures."
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Preparation of Deflavo-enzymes and Reconstitution with
Flavins--
To examine the effect of 6-hydroxy-FAD on the activity of
the flavin domain, the deflavo-domains from the two CDHs were prepared, and their cellobiose-oxidizing activity was determined after
reconstitution with FAD or 6-hydroxy-FAD. The use of the flavin domains
prepared after limited proteolysis was unsuccessful. However, active
flavin domains were successfully obtained by reconstitution of whole deflavo-CDHs with flavin followed by limited proteolysis of the reconstituted CDHs. The deflavo-CDHs from P. chrysosporium
and H. insolens were prepared by incubation of the enzyme
with a high concentration of CaCl2 in the presence of
cellobiose under the different conditions, as described under
"Experimental Procedures."
The absorption spectra of the deflavo and reconstituted flavin domains
are shown in Fig. 9. The absorption
spectrum of the domain from P. chrysosporium CDH
reconstituted with FAD (Fig. 9A) was almost identical with
that of the native domain, whereas that of the domain from H. insolens CDH reconstituted with 6-hydroxy-FAD CDH (Fig.
9B) was similar to but not identical with that of native domain, which contains a mixture of FAD and 6-hydroxy-FAD, as shown in
the previous section. Compared with the spectrum of the native flavin
domain (Fig. 7A), reconstitution with 6-hydroxy-FAD caused
loss of the shoulder peak around 470 nm, but the long-wavelength absorbance peak of 6-hydroxy-FAD shifted to 660 nm after binding to the
protein, as in the native sample.

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Fig. 9.
Absorption spectra of oxidized and reduced
flavin domains reconstituted with FAD or 6-hydroxy-FAD and their
deflavo-flavin domains. The flavin domain reconstituted with FAD
(oxidized, ; reduced, · · ·) or 6-hydroxy-FAD
(oxidized,   ; reduced, - - - ) and the deflavo samples
(. . . . . . . . . . .) were prepared as described under
"Experimental Procedures," and the spectra were recorded in 20 mM HEPES buffer, pH 7.5. Reduced spectra of flavin domains
were obtained by addition of 50 µM cellobiose.
A, experiments with flavin domain of P. chrysosporium CDH; B, experiments with flavin domain of
H. insolens CDH.
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Cellobiose-oxidizing activity of the enzymes with reconstituted FAD or
6-hydroxy-FAD is shown in Table I. The
activity of the P. chrysosporium domain reconstituted with
FAD was comparable with that of the native sample, indicating that the
reconstitution procedure had been successful. The activity of the
flavin domains reconstituted with 6-hydroxy-FAD from P. chrysosporium and H. insolens amounted to about
one-third at pH 4.0 and less than one-sixth at pH 7.5 as compared with
that of the domains reconstituted with FAD. The activity of the
H. insolens enzyme reconstituted with FAD was 1.8-fold
higher than that of the native enzyme at pH 7.5, whereas that of the
enzyme reconstituted with 6-hydroxy-FAD was about 60% of the native
one, a finding which can be well explained by the fact that the native
enzyme contains a mixture of 60% 6-hydroxy-FAD and 40% FAD.
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Table I
Effect of reconstitution for specific activity (s 1) of
cellobiose oxidizing activity
Cellobiose oxidation was monitored by reduction of ubiquinone as
described under "Experimental Procedures." ND, not detected.
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Thus, these spectrophotometric and activity measurements of the
reconstituted enzyme confirmed that CDH from H. insolens
contains 6-hydroxy-FAD as the dominant cofactor that is catalytically
active, though less than FAD. It is also clear that the pH dependence of the activity was not because of the flavin cofactor but, rather, was
because of the protein molecule because the pH dependence of the two
CDHs was little affected by flavin replacement, as judged from the
activities at pH 4.0 and pH 7.5.
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DISCUSSION |
Three CDH fractions were isolated from H. insolens
crude cellulase, whereas only one CDH was contained in culture solution of P. chrysosporium. Two fractions containing 92 and 94 kDa
species were separated from H. insolens by anion exchange
chromatography, as reported by Schou et al. (19), but one of
them (92 kDa) was further separated into two fractions by hydrophobic
chromatography using phenyl-Toyopearl 650M in this experiment although
these two minor CDH species had the same molecular mass (92 kDa), the same isoelectric point (4.0), and similar absorption spectra. It is not
clear at the moment whether these fractions are the different gene
products or products of post-translational modification. In this study,
the major fraction of CDH (94 kDa, pI 4.4) was used for experiments as
CDH from H. insolens.
The optimum pH of the cellobiose-cytochrome c oxidoreductase
activity of CDH from P. chrysosporium was in the acidic pH
range, whereas that of CDH from H. insolens was at neutral
pH. This is consistent with the pH optima for cellulase activity and
fungal growth on cellulose, which are pH 3-5 and 7-8 for P. chrysosporium and H. insolens, respectively (25, 26).
In this way CDH can cooperate effectively with cellulase in the
cellulose biodegradation process in both fungi. The absorption spectrum
of the oxidized forms of two CDHs were typical of hemoproteins though
there were small differences between two spectra. To clarify these
phenomena, limited proteolysis with papain was performed to prepare
heme- and flavin-containing domains. Like P. chrysosporium
CDH (11), H. insolens CDH was cleaved into two domains by
papain. In both CDHs, the sum of the molecular weights of the two
domains was almost identical to the molecular weight of the native CDH,
suggesting that papain cleaved a single site between the two domains,
although the cleavage site in H. insolens CDH was different
from that in P. chrysosporium CDH, resulting in a smaller
molecular weight of the heme domain and a larger molecular weight of
the flavin domain.
The absorption spectrum and midpoint potential were measured to
characterize the heme domain. Both heme domains showed typical b-type
heme spectra, and no difference was found in either the oxidized or the
reduced spectra. Moreover, they showed almost the same pH dependence of
midpoint potential. Although Schou et al. suggested that a
difference in the environment of heme causes the difference of optimum
pH between P. chrysosporium and H. insolens CDH
(19), the present experiments clearly demonstrate that the difference
in pH dependence is not because of heme as a prosthetic group but
rather is because of the flavin domain. The profile of pH dependence of
cellobiose-oxidizing activity was very similar for the flavin domain
and intact CDH in both cases, indicating that the optimum pH of CDH
mostly depends on the activity of the flavin domain.
The prepared flavin domain of H. insolens CDH showed a green
color with a broad peak at long wavelength, whereas that of P. chrysosporium CDH was yellow, showing a typical riboflavin-type spectrum. A similar spectrum to that of the flavin domain of H. insolens CDH has been reported by Mayhew et al. for the
green chromophore, 6-hydroxy-FAD, in electron-transferring flavoprotein (ETF) from Peptostreptococcus elsdenii (27). Upon binding to the CDH flavin domain, free 6-hydroxy-FAD showed a shift of the long-wavelength absorption from 600 to 660 nm, as in the case of ETF.
HPLC analysis of extracted flavin was carried out to identify and
quantify the prosthetic group of the flavin domain. Comparison with
authentic samples indicated that the flavin cofactor of H. insolens CDH is a mixture of 60% 6-hydroxy-FAD and 40% FAD,
whereas that of P. chrysosporium CDH consisted only of FAD.
This finding accounts well for the existence of shoulders at 380 and
475 nm in the absorption spectrum of H. insolens flavin
domain (Fig. 7A). Morpeth and Jones isolated cellobiose
quinone dehydrogenase (CBQ) as a simple flavoprotein from P. chrysosporium and showed that it has a nonfluorescent green
chromophore as the active cofactor (28). The spectrum of their native
enzyme suggested that 6-hydroxy-FAD was present as a minor component of
the prosthetic group. However, in the case of H. insolens
CDH, 6-hydroxy-FAD was contained as the dominant cofactor. The spectral
patterns of the other minor fractions of CDH from H. insolens were similar to that of the major fraction, suggesting
that all CDHs from H. insolens have the 6-hydroxy flavin
chromophore. Thus, H. insolens CDH is unique in containing
6-hydroxy-FAD as the dominant cofactor. As the activity of CDH
containing 6-hydroxy-FAD was lower than that of the enzyme reconstituted with normal FAD, the physiological significance of the
presence of 6-hydroxy-FAD is not clear but may reflect biosynthetic factors.
From the reconstitution experiments with 6-hydroxy-FAD or FAD, it is
clear that the pH dependence was not because of the flavin cofactor but
was intrinsic to the protein molecule. Reconstitution with FAD enhanced
the cellobiose-oxidizing activity of H. insolens flavin
domain, indicating that CDH containing 6-hydroxy-FAD is active but has
a lower cellobiose-oxidizing activity than the enzyme containing normal
flavin. Systematic studies using various modified flavins with
different redox potentials will be needed to see whether potential
change of the prosthetic group affects the activity because the
midpoint potential of FAD (
219 mV; Ref. 29) is more positive than
that of 6-hydroxy-FAD (
255 mV; Ref. 30), and such a study is in progress.