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Journal of Bacteriology, November 1998, p. 5646-5651, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Regio- and Stereospecific Conversion of
4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol
Oxidase
Robert H. H.
van den
Heuvel,
Marco W.
Fraaije,
Colja
Laane, and
Willem J. H.
van Berkel*
Laboratory of Biochemistry, Department of
Biomolecular Sciences, Wageningen University Research Center,
Wageningen, The Netherlands
Received 22 June 1998/Accepted 31 August 1998
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ABSTRACT |
The regio- and stereospecific conversion of prochiral
4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and
4-n-propylphenols. These short-chain 4-alkylphenols are
stereoselectively hydroxylated to the corresponding
(R)-1-(4'-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and
M. C. R. Franssen, Biotechnol. Bioeng. 59:171-177, 1998).
(S)-1-(4'-Hydroxyphenyl)ethanol was found to be a far
better substrate than (R)-1-(4'-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly
no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were
exclusively converted by vanillyl-alcohol oxidase to the corresponding
1-(4'-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature
of the alkyl side chain. The enzymatic conversion of
4-sec-butylphenol resulted in two
(4'-hydroxyphenyl)-sec-butene isomers with identical masses
but different fragmentation patterns. We conclude that the water
accessibility of the enzyme active site and the orientation of the
hydrophobic alkyl side chain of the substrate are of major importance
in determining the regiospecific and stereochemical outcome of
vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.
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INTRODUCTION |
Vanillyl-alcohol oxidase (EC
1.1.3.38) is a flavoprotein from Penicillium simplicissimum
that originally was shown to catalyze the oxidation of vanillyl alcohol
to vanillin with the simultaneous reduction of molecular oxygen to
hydrogen peroxide (2). The biological function of
vanillyl-alcohol oxidase is unknown, but recent studies have indicated
that the enzyme is involved in the biodegradation of
4-(methoxymethyl)phenol (6). Vanillyl-alcohol oxidase is a
homooctamer, with each 64-kDa subunit containing an
8
-(N3-histidyl)-flavin adenine dinucleotide
(FAD) as a covalently bound prosthetic group (2). The
vaoA gene has been cloned (1), and the
vanillyl-alcohol oxidase structure has been determined at 2.5-Å
resolution (11). These studies, together with sequence alignments, have revealed that the enzyme belongs to a novel
oxidoreductase family sharing a conserved FAD binding domain
(8). Vanillyl-alcohol oxidase is a versatile biocatalyst,
mechanistically. It can convert a wide range of 4-hydroxybenzylic
compounds by catalyzing oxidation, hydroxylation, demethylation,
deamination, and desaturation reactions (3, 5). Some of
these reactions are of particular interest for biotechnological
applications (16).
Based on studies with eugenol and 4-(methoxymethyl)phenol, we have
proposed that vanillyl-alcohol oxidase catalysis involves the initial
transfer of a hydride from the C- atom of the substrate to N-5 of
the flavin cofactor (5, 7). Formation of the resulting p-quinone methide intermediate is facilitated by substrate
deprotonation upon binding. The p-quinone methide product
intermediate subsequently reacts with water in the enzyme active site
to form the final aromatic product (Fig.
1).A similar reaction mechanism has been proposed for the conversion of
4-alkylphenols by the related bacterial flavocytochrome
p-cresol methylhydroxylase (10, 13). Further support for the hydride transfer mechanism comes from crystallographic data (11). The vanillyl-alcohol oxidase structure has
revealed that the active site is located in the interior of the protein and contains an anionic binding pocket facilitating substrate deprotonation. His422 was identified as the residue which covalently links the flavin cofactor. The crystal structure also revealed that the
side chain of Asp170 is located close to the C- atom of the
substrate. However, the exact function of this residue in catalysis
remains to be elucidated.
Vanillyl-alcohol oxidase displays a remarkable reactivity towards
short-chain 4-alkylphenols. Recent studies showed that 4-ethylphenol and 4-n-propylphenols are stereospecifically converted into
the corresponding 1-(4'-hydroxyphenyl)alcohols with an e.e
of 94% for the R enantiomers. During these reactions,
considerable amounts of 1-(4'-hydroxyphenyl)alkenes are formed as side
products, indicating that rearrangement of the p-quinone
methide intermediate competes with water addition (3).
4-Methylphenol, the parent substrate of p-cresol
methylhydroxylase, is a very poor substrate for vanillyl-alcohol oxidase. Crystallographic and kinetic data suggest that this is due to
the stabilization of a flavin N-5 adduct (9, 11). To obtain
more insight into the catalytic performance of this unusual
flavoenzyme, we have addressed the reactivity and stereochemistry of
vanillyl-alcohol oxidase with medium-chain 4-alkylphenols, bicyclic
phenols, and 4-hydroxyphenyl alcohols. The results of this study are
discussed in relation to the recently determined crystal structure.
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MATERIALS AND METHODS |
Chemicals.
4-Methylphenol, 4-ethylphenol,
4-n-propylphenol,
2-methoxy-4-n-propylphenol, 4-isopropylphenol,
4-n-butylphenol, 4-sec-butylphenol, 4-(3'-methylcrotyl)phenol, 5-indanol,
5,6,7,8-tetrahydro-2-naphthol, vanillyl alcohol
(4-hydroxy-3-methoxybenzyl alcohol), vanillin (4-hydroxy-3-methoxybenzaldehyde), tyramine [4-(2-aminoethyl)phenol], trans-isoeugenol [1-(4'-hydroxy-3'-methoxyphenyl)propene],
3-(4'-hydroxyphenyl)propanol, 4-phenylphenol, and 4-benzylphenol were
from Aldrich. 4-n-Pentylphenol, 4-n-heptylphenol,
4-n-nonylphenol, 4-vinylphenol, and
2-(4'-hydroxyphenyl)ethanol were obtained from Lancaster. Frambinon
[1-(4'-hydroxyphenyl)-2-butanone] was from Quest.
4-(3'-Methylcrotyl)phenol (75% pure, based on gas chromatography-mass
spectrometry [GC-MS] analysis) was purified to apparent
homogeneity
by high-performance liquid chromatography (HPLC) with
a Lichrospher
RP8 reverse-phase column. Racemic 1-(4'-hydroxyphenyl)ethanol
and
racemic 1-(4'-hydroxyphenyl)propanol were synthesized by
Drijfhout
et al. (
3).
(
R)-1-(4'-Hydroxyphenyl)ethanol was obtained from
the
enzymatic conversion of 4-ethylphenol (
3).
Enzyme purification.
Vanillyl-alcohol oxidase was purified
from P. simplicissimum (Oudem.) Thom. CBS 170.90 (ATCC
90172) as described previously (5).
Analytical methods.
All experiments were performed in
air-saturated 50 mM potassium phosphate buffer, pH 7.5, at 25°C,
unless stated otherwise. Enzyme concentrations were measured
spectrophotometrically by using a molar absorption coefficient,
439 of 12.5 mM
1 cm1 for
protein-bound FAD (2). Vanillyl-alcohol oxidase activity was
determined by monitoring absorption spectrum changes of aromatic substrates or by oxygen consumption experiments using a Clark electrode. Vanillin production was measured at 340 nm
(340 = 15.0 mM1 cm1).
Formation of 4-vinylphenol and 1-(4'-hydroxyphenyl)ethanol from
4-ethylphenol was measured at 255 nm (255 = 14.3 mM1 cm1) and 270 nm (270 = 1.2 mM1 cm1), respectively. Formation of
1-(4'-hydroxyphenyl)propanol was monitored at 270 nm
(270 = 1.3 mM1 cm1).
Absorption spectra were recorded on an SLM Aminco DW-2000
spectrophotometer. Dissociation constants of enzyme-inhibitor complexes
were determined from flavin absorption perturbation difference spectra
by titration of a known concentration of enzyme with the inhibitor.
Product identification.
HPLC experiments were performed with
an Applied Biosystems 400 pump equipped with a Waters 996 photodiode
array detector. Enzyme reaction products were separated with a 4.6- by
150-mm Lichrospher RP8 column and isocratic methanol-water mixtures
containing 1% acetic acid as the eluent. The methanol/water ratio
depended on the type of aromatic compounds to be separated. For
resolution of cis and trans isomers, a 4.6- by
100-mm Microspher C18 column was used under conditions
similar to those used for the Lichrospher RP8 column. Relative yields
of aromatic products were determined by the molar absorption
coefficients of 4-vinylphenol (265 = 13.0 mM1 cm1), 1-(4'-hydroxyphenyl)ethanol
(265 = 0.9 mM1 cm1),
1-(4'-hydroxyphenyl)propanol (265 = 1.0 mM1 cm1), 4-hydroxyacetophenone
(265 = 12.5 mM1 cm1), and
4-hydroxypropiophenone (265 = 12.9 mM1
cm1) in the appropriate HPLC solvent.
GC-MS analysis was performed on a Hewlett-Packard HP 6090 gas
chromatograph and an HP 5973 mass spectrometer equipped with
an HP-5
column. Reaction mixtures, containing 0.5 to 1.0 mM aromatic
substrate
and 0.2 to 0.8 µM enzyme, were incubated at 25°C until
the reaction
ceased. Samples prepared by extracting the reaction
mixtures with 2 volumes of diethylether were injected without
derivatization. The
temperature program was 5 min isothermal at
50°C, followed by an
increase to 240°C at 7°C min
1. Relative yields were
calculated from the integration of the
total ion current peak areas.
1H-nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker AMX 400-MHz spectrometer at 24°C. Samples were prepared
by freeze-drying the isolated products obtained from HPLC separations
and dissolving the products in CD
3OD or D
2O.
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RESULTS |
Substrate specificity.
Earlier studies revealed that
vanillyl-alcohol oxidase has a relaxed substrate specificity towards
4-hydroxybenzylic compounds (3, 5) (Fig.
2). 4-Ethylphenol,
4-n-propylphenol, and 2-methoxy-4-n-propylphenol are efficiently oxidized by vanillyl-alcohol oxidase (9). In this study, we investigated the substrate specificity in further detail. Medium-chain 4-alkylphenols with aliphatic side chains of up to
seven carbon atoms were converted by vanillyl-alcohol oxidase.
4-Alkylphenols with longer aliphatic side chains, like 4-n-nonylphenol, did not react. Table
1 shows that straight-chain 4-alkylphenols interact tightly with vanillyl-alcohol oxidase and that
the catalytic efficiency (expressed as
kcat/Km) is optimal at an
alkyl chain length of three carbon atoms. Substituents in the aliphatic
side chain significantly affected the catalytic efficiency of
vanillyl-alcohol oxidase. Like 4-isopropylphenol and
4-sec-butylphenol, frambinon
[1-(4'-hydroxyphenyl)-2-butanone] was converted at a significant rate
(Table 1). As 4-allylphenols are among the best substrates of
vanillyl-alcohol oxidase (5), it was of interest to study
the reactivity of 4-(3'-methylcrotyl)phenol. Table 1 shows that this
branched-chain 4-allylphenol is indeed a good substrate.
Vanillyl-alcohol oxidase was also active with bicyclic phenols. Both
5,6,7,8-tetrahydro-2-naphthol and 5-indanol were readily converted
(Table 1). In contrast, no activity was found with 4-benzylphenol.
In an earlier study, we reported that 1-(4'-hydroxyphenyl)alcohols,
produced from the enzymatic conversion of short-chain
4-alkylphenols,
are not readily oxidized to the corresponding
alkanones. This might be
due to the high enantioselectivity of
the initial hydroxylation
reaction, which predominantly yields
the
R isomer
(
3). Therefore, it was of interest to study the
enzymatic
conversion of pure enantiomers. Table
1 shows that
(
S)-1-(4'-hydroxyphenyl)ethanol is a far better substrate
than
(
R)-1-(4'-hydroxyphenyl)ethanol. This explains why in
the reaction
of vanillyl-alcohol oxidase with 4-ethylphenol, hardly any
1-(4'-hydroxyphenyl)acetophenone
is formed. For the enzymatic
conversion of 4-
n-propylphenol and
2-methoxy-4-
n-propylphenol, the
R isomer of the
corresponding
aromatic alcohol was identified as the main product as
well (
3).
This suggests that for these substrates, the same
kinetic resolution
mechanism is operative. Vanillyl-alcohol oxidase
reacted poorly
with C-
and C-
hydroxylated 4-hydroxyphenyl
alcohols (Table
1). In line with this, the enzyme was not active with
4-(aminoalkyl)phenols
including tyramine.
Product identification.
Vanillyl-alcohol oxidase produces
different amounts of 1-(4'-hydroxyphenyl)alcohols and
1-(4'-hydroxyphenyl)alkenes from short-chain 4-alkylphenols
(3). HPLC analysis of the enzymatic conversion of
4-n-propylphenol showed that the relative yield of
1-(4'-hydroxyphenyl)propene was 32% (Table
2). Further examination of this fraction
on a Microspher C18 column revealed two peaks with
comparable peak areas near the expected elution time of the aromatic
alkene (Fig. 3A). The two products showed
slightly different absorption characteristics, with maxima at 251 and
256 nm, respectively, and minor differences in the characteristic
aromatic alkene shoulder near 290 nm (Fig. 3B). The mass spectra of the
products were identical, with a molecular ion at m/z
(relative intensity) (M)+ 134 (100%) and the following
diagnostic fragments with more than 25% abundance: 132 (77%) and 107 (30%). These results indicate that a mixture of cis-trans
isomers of 1-(4'-hydroxyphenyl)propene is formed during the
vanillyl-alcohol oxidase-mediated conversion of
4-n-propylphenol (Table 3).
The vanillyl-alcohol oxidase catalyzed conversion of
2-methoxy-4-n-propylphenol predominantly yielded the
alcoholic product (Table 2). Moreover, from the identical HPLC elution
time and absorption spectrum of the main alkenylic product and the
reference compound trans-isoeugenol, it is evident that
trans-isoeugenol is formed in large excess over the
cis isomer (Table 3).
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TABLE 2.
Relative yields of products formed from the conversion of
4-alkylphenols by vanillyl-alcohol oxidase
from P. simplicissimum
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FIG. 3.
(A) HPLC analysis of aromatic products formed from the
conversion of 4-n-propylphenol by vanillyl-alcohol oxidase
from P. simplicissimum. The elution solvent was
methanol-water-acetic acid (50:50:1). (B) Absorption spectrum of
cis-1-(4'-hydroxyphenyl)propene and
trans-1-(4'-hydroxyphenyl)propene.
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TABLE 3.
Relative yields of cis and trans
isomers of 4-hydroxyphenyl alkenes formed from the conversion of
4-alkylphenols by vanillyl-alcohol oxidase
from P. simplicissimum
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HPLC analysis of the enzymatic conversion of
4-
sec-butylphenol revealed a low yield of the alcoholic
product (Table
2). In
addition to
2-(4'-hydroxyphenyl)-
sec-butanol, two aromatic butenes
were
produced. However, these two products were not
cis-trans isomers, because their mass spectra showed different fragmentation
patterns. The first eluted aromatic butene contained a molecular
ion at
m/z (relative intensity) (M)
+ 148 (96%) and the
following diagnostic fragments with more than
25% abundance: 147 (35%), 133 (100%), and 105 (37%). This fragmentation
pattern is
indicative of 2-(4'-hydroxyphenyl)-
sec-butene (Fig.
4A). The second alkenylic product
contained a molecular ion at
m/z (relative intensity)
(M)
+ 148 (100%) and the following diagnostic fragments
with more than
25% abundance: 147 (26%), 133 (76%), 119 (88%), and
91 (38%).
This points to the production of
1-(4'-hydroxyphenyl)-
sec-butene
(Fig.
4B). Furthermore,
comparisons of the absorption spectrum
of
2-(4'-hydroxyphenyl)-
sec-butene with identified
cis-trans isomers
of medium-chain
1-(4'-hydroxyphenyl)alkenes (see below) indicate
that only
cis-2-(4'-hydroxyphenyl)-
sec-butene is formed.
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FIG. 4.
Mass-spectral analysis of aromatic butenes formed from
the conversion of 4-sec-butylphenol by vanillyl-alcohol
oxidase from P. simplicissimum. (A)
cis-2-(4'-Hydroxyphenyl)-sec-butene. (B)
1-(4'-Hydroxyphenyl)-sec-butene.
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No alcoholic products are formed in the reaction of vanillyl-alcohol
oxidase with aromatic substrates having an alkyl side
chain of at least
four carbon atoms (Table
2). With 4-
n-butylphenol,
mostly
one 1-(4'-hydroxyphenyl)butene isomer was formed. The
1H-NMR spectrum of this compound showed vinylic bands at
5.52 ppm
(dt,
J = 11.5 Hz, 7.0 Hz, 1H) and 6.30 ppm (d,
J = 11.5 Hz, 1H)
for the C-
proton and C-
proton,
respectively. This established
that the aromatic butene has a
cis-relative stereochemistry (Table
3). HPLC analysis of the
enzymatic conversion of 4-
n-pentylphenol
showed a mixture of
two alkenylic products. The mass spectra of
the compounds were
identical, with a molecular ion at
m/z (relative
intensity)
(M)
+ 162 (30%) and the following diagnostic fragments with
more than
25% abundance: 133 (100%) and 105 (27%). The two products
had
slightly different absorption characteristics with respect to
the
absorption maximum and the aromatic alkene shoulder (not shown).
These
results point to the formation of a mixture of
cis- and
trans-1-(4'-hydroxyphenyl)pentene.
1H-NMR
analysis revealed differences between the two compounds
concerning the
coupling constants of the vinylic protons:
trans isomer,
6.03 ppm (dt,
J = 15.8 Hz, 7.1 Hz, 1H) and 6.27 ppm (d,
J = 15.8 Hz, 1H);
cis isomer, 5.49 ppm (dt,
J = 11.2 Hz, 7.1 Hz,
1H) and 6.29 ppm (d,
J = 11.2 Hz, 1H) (Fig.
5). In order to rule
out possible
cis-trans isomerization in the enzyme active site,
the
isolated isomers of 1-(4'-hydroxyphenyl)pentene were incubated
with
vanillyl-alcohol oxidase in 50 mM potassium phosphate buffer,
pH 7.5. HPLC analysis clearly demonstrated that the configurations
of the
cis and
trans isomers of
1-(4'-hydroxyphenyl)pentene did
not change with time. In analogy to the
reaction of 4-
n-pentylphenol,
enzymatic conversion of
4-
n-heptylphenol resulted in the formation
of equal amounts
of the
cis and
trans isomers of
1-(4'-hydroxyphenyl)heptene
(Table
3).
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FIG. 5.
Expanded 1H-NMR spectra of aromatic products
formed from the conversion of 4-n-pentylphenol by
vanillyl-alcohol oxidase from P. simplicissimum. (A)
cis-1-(4'-Hydroxyphenyl)pentene. (B)
trans-1-(4'-Hydroxyphenyl)pentene.
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Conversion of eugenol by vanillyl-alcohol oxidase results in the
stoichiometric formation of coniferyl alcohol (
5).
Interestingly,
enzymatic oxidation of 4-(3'-methylcrotyl)phenol
resulted in the
formation of two aromatic products (Table
2). The mass
spectrum
of the most polar product contained a molecular ion at
m/z (relative
intensity) (M)
+ 178 (24%) and the
following diagnostic fragments with more than
25% abundance: 163 (71%), 145 (63%), 127 (37%), 115 (26%), 107
(100%), and 43 (47%). This points towards the formation of the
C-
hydroxylated product 4-(3'-methyl-1'-butene-3'-ol)phenol
(Fig.
6A). Further evidence for the
structure of the product was obtained
by
1H-NMR analysis.
The
1H-NMR spectrum in D
2O gave bands at
1.80 (s, 6H, methyl), 6.20
(d,
J = 16.4 Hz, 1H,
vinylic), 6.43 (d,
J = 16.4 Hz, 1H, vinylic),
6.77 (d,
J = 8.4 Hz, 2H, aromatic), and 7.28 (d,
J = 8.4 Hz, 2H,
aromatic). The coupling constants of
the vinylic protons also
established that the product has a
trans-relative stereochemistry.
Besides this alcoholic
compound a second, more hydrophobic product
was formed with a molecular
ion at
m/z (relative intensity) (M)
+ 160 (46%)
and the following diagnostic fragments with more than
25% abundance:
145 (100%), 127 (44%), and 115 (35%) (Fig.
6B).
The
1H-NMR spectrum in D
2O gave bands at
1.85 (3H, s, methyl), 4.99
(d,
J = 2.0 Hz, 1H, vinylic),
5.03 (d,
J = 2.1 Hz, 1H, vinylic),
6.52 (d,
J = 16.6 Hz, 1H, vinylic), 6.78 (d,
J = 8.5 Hz, 2H, aromatic),
6.81 (d,
J = 16.6 Hz, 1H, vinylic), and 7.34 (d,
J = 8.5 Hz, 2H,
aromatic). The above data are
consistent with the second product
being
4-(3'-methylbutadiene)phenol. Furthermore, the coupling
constants
of the vinylic protons established that the C-
and
C-
protons
have a
trans-relative stereochemistry.
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FIG. 6.
Mass-spectral analysis of aromatic products formed from
the conversion of 4-(3'-methylcrotyl)phenol by vanillyl-alcohol oxidase
from P. simplicissimum. (A)
4-(3'-Methyl-1'-butene-3'-ol)phenol. (B)
4-(3'-Methylbutadiene)phenol.
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Substantial differences in the ratios of aromatic products were
observed when vanillyl-alcohol oxidase was incubated with
bicyclic
phenols. With 5-indanol, the initial alcoholic product
was readily
further oxidized to the alkanone, and only small amounts
of the
alkenylic product were formed (Table
2). However, with
5,6,7,8-tetrahydro-2-naphthol, dehydrogenation of the putative
p-quinone methide product intermediate was clearly favored
over
water addition (Table
2). Because the bicyclic products were
rather unstable, no attempt was made to study their identity in
further
detail.
Product inhibition.
The enzymatic conversion of 4-alkylphenols
revealed product inhibition, particularly with medium-chain
4-alkylphenols. This is in agreement with the earlier observation that
the alkenylic compounds coniferyl alcohol and isoeugenol are strong
competitive inhibitors of vanillyl-alcohol oxidase (5).
4-Vinylphenol, one of the products formed from the enzymatic conversion
of 4-ethylphenol (3), appeared to be a very strong
competitive inhibitor for vanillyl-alcohol oxidase
(Ki = 3 ± 1 µM [Fig.
7]). Similar inhibition during the
conversion of 4-ethylphenol was reported for p-cresol methylhydroxylase (13). No inhibition constants of other
aromatic alkenes were determined. However, the product inhibition
observed during the enzymatic conversion of medium-chain 4-alkylphenols suggests that 4-alkenylphenols interact tightly with the enzyme. This
is supported by the crystal structures of vanillyl-alcohol oxidase in
complex with isoeugenol and 1-(4'-hydroxyphenyl)heptene (11).
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FIG. 7.
Competitive inhibition of vanillyl-alcohol oxidase by
4-vinylphenol. The enzymatic conversion of vanillyl-alcohol at pH 7.5, 25°C, was measured by the increase in absorbance at 340 nm. The
presence of the following are indicated: no inhibitor ( ); 10 µM
4-vinylphenol ( ); 20 µM 4-vinylphenol ( ).
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DISCUSSION |
In this study, we have described the reactivity of the covalent
flavoprotein vanillyl-alcohol oxidase with medium-chain 4-alkylphenols, bicyclic phenols, and 4-hydroxyphenyl alcohols. The enzyme was active,
with 4-alkylphenols bearing aliphatic side chains of up to seven carbon
atoms. This agrees perfectly with structural data which showed that the
active-site cavity of vanillyl-alcohol oxidase is completely filled
upon binding of the inhibitor 1-(4'-hydroxyphenyl)heptene (11).
During the conversion of short-chain 4-alkylphenols by vanillyl-alcohol
oxidase, (R)-1-(4'-hydroxyphenyl)alcohols are formed as
major products (3). In this paper, clear evidence is
provided that the low yield of 1-(4'-hydroxyphenyl)alkanones is due to a preferred stereospecific oxidation of
(S)-1-(4'-hydroxyphenyl)alcohols (Fig.
8). The low catalytic efficiency of the
conversion of the R isomers suggests that the binding of
1-(4'-hydroxyphenyl)alcohols is energetically unfavored when the C-
hydroxyl group of the substrate faces the flavin ring. Interestingly,
differences in the stereospecificities of oxidation of
1-(4'-hydroxyphenyl)alcohols were reported for the related
flavocytochromes 4-ethylphenol methylenehydroxylase (15) and
p-cresol methylhydroxylase (12).
Besides catalyzing stereoselective hydroxylation reactions,
vanillyl-alcohol oxidase is regioselective as well. Whereas
4-alkylphenols are exclusively hydroxylated at the C- atom,
4-allylphenols, like eugenol, are hydroxylated at the C- atom
(5). These differences in regioselective hydroxylation
suggest that the site of water attack is dependent on the
delocalization of charge in the enzyme-bound p-quinone
methide product intermediate. As noted before (11), Asp170
might activate the water, thereby acting as an active-site base.
Interestingly, the eugenol derivative 4-(3'-methylcrotyl)phenol is not
exclusively hydroxylated at the C- position. With this 4-allylphenol, dehydrogenation at the C- position occurs to a significant extent.
In contrast to short-chain 4-alkylphenols, medium-chain 4-alkylphenols
are exclusively converted by vanillyl-alcohol oxidase to the
corresponding 1-(4'-hydroxyphenyl)alkenes. This points to rearrangement
of the p-quinone methide product intermediate and suggests
that the efficiency of water addition to this highly reactive
electrophilic species is dependent on the water accessibility of the
enzyme active site. The presence of reduced glutathione during turnover
of vanillyl-alcohol oxidase with medium-chain 4-alkylphenols did not
influence the stoichiometric formation of the alkenylic products,
indicating that rearrangement of the p-quinone methide
intermediate occurs in the enzyme active site. Based on the crystal
structures of vanillyl-alcohol oxidase-inhibitor complexes
(11), we assume that no significant conformational changes
are induced upon binding of the more bulky 4-alkylphenols. Moreover,
the crystallographic data show that the carboxylate oxygen atoms of
Asp170 are located at about 3.5 Å from the C- atom of the
inhibitors (11). Therefore, rearrangement of the p-quinone methide intermediate might be induced by proton
abstraction by Asp170, again acting as an active-site base.
In this paper, we have demonstrated that vanillyl-alcohol oxidase
dehydrogenates medium-chain 4-alkylphenols stereospecifically. This
suggests that the p-quinone methide intermediates formed with these substrates are rigidly bound in a specific orientation in
the enzyme active site. This cis-trans stereospecificity is not unique among flavoenzymes. For example, acyl-coenzyme A
dehydrogenases introduce a trans double bond between C-2 and
C-3 of their coenzyme A substrates (14), whereas glycolate
oxidase shows specificity for re hydrogen abstraction when
prochiral glycolate is used as a substrate (4). In contrast
to these enzymes, the relative cis-trans stereochemistry of
vanillyl-alcohol oxidase is strongly dependent on the nature of the
alkyl side chain of the substrate. However, no correlation was found
between the stereochemical preference and the bulkiness or length of
the 4-alkylphenol side chains.
In summary, the results presented here show that vanillyl-alcohol
oxidase is active with a wide range of 4-alkylphenols. Short-chain 4-alkylphenols are mainly hydroxylated to aromatic alcohols, whereas medium-chain 4-alkylphenols are exclusively dehydrogenated to aromatic
alkenes. We conclude that the regio- and stereospecificity of the
vanillyl-alcohol oxidase-mediated reactions is mainly determined by (i)
the intrinsic reactivity of the enzyme-bound p-quinone methide intermediate, (ii) the water accessibility of the enzyme active
site, and (iii) the orientation of the hydrophobic alkyl side chain of
the substrate.
| |
ACKNOWLEDGMENT |
This research was performed within the framework of the
Innovation Oriented Research Program (IOP) Catalysis of the Dutch Ministry of Economy Affairs (project IKA96005).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biomolecular Sciences, Laboratory of Biochemistry, Wageningen
University Research Center, Dreijenlaan 3, 6703 HA Wageningen, The
Netherlands. Phone: 31-317-482861. Fax: 31-317-484801. E-mail:
willem.vanberkel{at}fad.bc.wau.nl.
| |
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Abstract
Introduction
