Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-: Trans -stilbene and stilbenoids modification by fungal peroxygenases

Article abstract of DOI:10.1039/c8cy00272j

This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H₂O₂ as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200000) than MroUPO (26000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (k_cat/K_m) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of ¹⁸O from H₂¹⁸O₂ into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV.

Full text of DOI:10.1039/c8cy00272j

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Selective synthesis of the resveratrol analogue
4
,4′-dihydroxy-trans-stilbene and stilbenoids
Cite this: DOI: 10.1039/c8cy00272j
modification by fungal peroxygenases†
a
b
c
c
a
Carmen Aranda, René Ullrich, Jan Kiebist, Katrin Scheibner, José C. del Río,
b
d
a
Martin Hofrichter, Angel T. Martínez
and Ana Gutiérrez
*
This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe
aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the
regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) ana-
logue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly
complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using
H O
2 2
as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000)
than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most effi-
cient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/K ) one and two orders of
m
magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best
UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium
globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the
trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as
substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin.
Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by
three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of
products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demon-
18
18
strated by incorporation of O from H
2 2
O into the stilbene hydroxylation products. Differences in the
number of phenylalanine residues at the heme access channels seems related to differences in aromatic hy-
droxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The
only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal
side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydrox-
ylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemi-
cal synthesis for the production of DHS, RSV and oxyRSV.
Received 6th February 2018,
Accepted 27th March 2018
DOI: 10.1039/c8cy00272j
rsc.li/catalysis
number of structure–activity studies have been carried out
that have revealed the molecular determinants of RSV neces-
Introduction
Resveratrol (RSV, 3,5-4′-trihydroxy-trans-stilbene) has emerged
in recent years as a fascinating compound because of its wide
spectrum of biological effects including protection against
metabolic, cardiovascular and other age-related complications
sary for its biological effects, such as the hydroxyl group in
position 4′ of the aromatic ring, together with the trans-
2
conformation. This information was used for the synthesis of
RSV analogues such as 4,4′-dihydroxy-trans-stilbene (DHS)
1
such as neurodegeneration and cancer. Therefore, a large
with enhanced cytotoxic, anti-proliferative and anti-tumor
3
properties in in vitro experiments. These studies demon-
a
strated as well that both 4- and 4′-OH groups are essential for
inducing estrogen receptor down-regulation. DHS was identi-
fied for the first time among the metabolites of the
trans-stilbene excreted in the urine of guinea pigs, rabbits
and mice and many years later, the cytochrome P450 iso-
forms involved in the oxidation of trans-stilbene to DHS were
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes
1
b
0, E-41012 Seville, Spain. E-mail: anagu@irnase.csic.es
Department of Bio- and Environmental Sciences, TU Dresden, Markt 23, 02763
Zittau, Germany
c
JenaBios GmbH, Löbstedter Str. 80, 07749 Jena, Germany
4
d
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040
Madrid, Spain
identified. Shortly after, DHS was found as a phytoalexin in
the bark of Yucca periculosa. More recently, the effects of
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
5
c8cy00272j
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DHS on cancer invasion and metastasis were demonstrated Experimental
6
in vivo.
Enzymes
Usually, DHS can be prepared chemically via several syn-
theses routes including Perkin condensation. However, this
7
The MroUPO enzyme is a wild peroxygenase isolated from liq-
uid cultures of M. rotula DSM-25031 (a fungus deposited at the
German Collection of Microorganisms and Cell Cultures,
Braunschweig). MroUPO was purified by fast protein liquid
chromatography (FPLC) to apparent homogeneity, and revealed
a molecular mass of 32 kDa and an isoelectric point between
pH 5.0 and 5.3. The UV-visible spectrum of the enzyme showed
a characteristic maximum at 418 nm (Soret band of heme-
chemical synthesis involves a sequence of steps, toxic
chemicals, and transition metal catalysts with low yield in
DHS. With the purpose of meeting the growing demand for
stilbenoids, alternative and sustainable approaches for their
production are required. The use of enzymes for organic syn-
thesis of fine chemicals and pharmaceuticals has become a
powerful alternative to chemical catalysts. Replacing organic/
metal catalysts by biocatalysts has several advantages, the
most important being excellent regio- and stereo-selectivity,
fewer side products, and reduction in environmental impact.
Biocatalysts that preferably catalyze the transfer of an oxy-
gen atom from peroxide to substrates are known as
peroxygenases and are classified in a separate sub-subclass,
14
thiolate proteins). All media and columns used for enzyme
isolation were purchased from GE Healthcare Life Sciences.
The AaeUPO (A. aegerita isoform II, 46 kDa) is another
wild peroxygenase, which was isolated from cultures of A.
aegerita grown in soybean-peptone medium, and subse-
quently purified using a combination of Q-Sepharose and SP-
9
Sepharose and Mono-S ion-exchange chromatographic steps.
(EC.1.11.2), which was approved in 2011 and at present com-
rCciUPO corresponds to the protein model 7249 from the
sequenced C. cinerea genome available at the DOE JGI (http://
genome.jgi.doe.gov/Copci1). It was expressed in Aspergillus
oryzae (patent WO/2008/119780), purified using a combina-
tion of S-Sepharose and SP-Sepharose ion-exchange chroma-
tography, and provided by Novozymes A/S (Bagsvaerd, Den-
prises five members. Among them, the unspecific
peroxygenase (UPO, EC 1.11.2.1) is the most prominent one
because of its frequency in fungal organisms and promiscuity
for oxygen transfer reactions that make it highly attractive as
industrial biocatalysts.
8
The first enzyme of this type was discovered in the basid-
9
mark) as
a
protein preparation. The recombinant
iomycete Agrocybe aegerita. UPOs are able to catalyze reac-
1
0
peroxygenase is a glycoprotein with a molecular mass around
44 kDa, a typical UV-vis spectrum with the Soret band at 418
nm, and the ability to oxygenate different aromatic com-
pounds including veratryl alcohol with a specific activity of
tions formerly assigned only to P450s, using a similar active
1
1
site and reaction chemistry. However, unlike P450s that are
intracellular enzymes and whose activation usually requires
an auxiliary enzyme and a source of reducing power, UPOs
are secreted proteins, therefore far more stable, and only
−
1
approx. 100 U mg (measured as described below).
1
2
CglUPO (36 kDa) is a third wild peroxygenase recently iso-
lated from cultures of C. globosum DSM-62110 (German Col-
lection of Microorganisms and Cell cultures), and purified by
ammonium sulfate precipitation and successive FPLC using
Q-Sepharose FF (ion exchange), Superdex75 (size exclusion),
require H O for activation. Similar enzymes have also been
2
2
1
3
found in other basidiomycetes, such as Coprinellus radians
and Marasmius rotula and there are strong indications for
their widespread occurrence in the fungal kingdom. Over
one-hundred peroxygenase-type genes (encoding enzymes of
the heme-thiolate peroxidase superfamily) have been identi-
1
4
1
5
1
8
and Mono Q (ion exchange) columns. All chromatographic
steps were accomplished with an ÄKTA purifier FPLC system
(GE Healthcare).
1
6
fied during the analysis of 24 basidiomycete genomes in-
1
7
cluding Coprinopsis cinerea. The C. cinerea peroxygenase
has not been isolated to date from the fungus, but one of the
peroxygenase genes from its genome was heterologously
expressed by Novozymes A/S (Bagsvaerd, Denmark). Very re-
cently, another peroxygenase has been found in the cellulo-
The purified proteins were electrophoretically homoge-
nous, as shown by sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under denaturing conditions, al-
though some minor contaminant proteins were present in
CglUPO (ESI† Fig. S1).
1
8
lytic
ascomycete
Chaetomium
globosum.
These
One activity unit (U) is defined as the amount of enzyme
oxidizing 1 μmol of veratryl alcohol to veratraldehyde (ε
peroxygenases have been shown to catalyze numerous inter-
1
9
3
10
esting oxygenation reactions on aromatic compounds and
later, their ability to oxyfunctionalize diverse aliphatics in-
−
1
−1
9
300 M cm ) in 1 min at 24 °C, pH 7.0 (the optimum pH
2
0–23
for benzylic hydroxylation by AaeUPO and some other
cluding linear
strates such as steroids and secosteroids
strated expanding their biotechnological interest.
and cyclic alkanes as well as complex sub-
2
4
25,26
peroxygenases) after addition of 2.5 mM H O .
2
2
was demon-
Model compounds
In this work, the ability of non-recombinant (hereinafter wild)
peroxygenases from A. aegerita (AaeUPO), M. rotula (MroUPO)
and C. globosum (CglUPO), and recombinant peroxygenase from
C. cinerea (rCciUPO), to catalyze the hydroxylation of
trans-stilbene is shown. Additionally, the UPO-catalyzed hydrox-
ylation of pinosylvin (Pin, a stilbenoid present in the heartwood
of conifers of the family Pinaceae) and RSV was studied.
trans-Stilbene ([E]-1,2-diphenylethene) and some stilbenoids,
including Pin (3,5-dihydroxy-trans-stilbene) and RSV (3,5,4′-
trihydroxy-trans-stilbene) were tested as substrates of the
above UPOs. Additionally, 4-hydroxy-trans-stilbene (4HS), 4,4′-
dihydroxy-trans-stilbene (DHS), oxyresveratrol (oxyRSV,
3,5,2′,4′-tetrahydroxy-trans-stilbene) and trans-stilbene epoxide
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were used as standards in GC-MS and HPLC analyses. Chemi-
cal structures of these compounds are shown in ESI† (Fig.
S2).
This short reaction time was selected to: i) obtain maximal
turnover numbers; and ii) avoid further oxidation of the first
product 4HS enabling the separate analysis of both reactions.
All reactions were carried out in triplicate. Product quantifi-
cation was carried out by GC-MS using external standard
Enzyme reactions
m
curves, and kinetic parameters (kcat, K ) were obtained by
Enzymatic reactions were performed at 0.1 mM substrate
concentration (1 mL reaction volume) and 30 °C, in 50 mM
phosphate buffer (pH 7 was selected for comparison). Prior
to use, the substrate was dissolved in acetone and added to
the buffer to give a final acetone concentration in the reac-
tion of 20% (v/v) (resulting in substrate solubilization and
best reaction rate); reactions without acetone were also
performed. Ascorbic acid was added to the reaction mixture
fitting the data to the Michaelis–Menten equation using
SigmaPlot software (Systat Softwarwe Inc., San Jose, CA, USA).
For total turnover number (TTN) determination of
trans-stilbene oxidation, reactions were performed with
AaeUPO (13 nM), MroUPO (0.2 μM) and rCciUPO (1.2 μM)
and substrate concentration was increased up to 5 mM in the
presence of 20% (v/v) of acetone.
(
except for CglUPO reactions) to prevent further oxidation of
GC-MS analyses
hydroxylated aromatic (phenolic) products and subsequent
2
7
The analyses were performed with a Shimadzu GC-MS QP
radical coupling, the best results being obtained using
mM concentration. Enzyme and H concentration were
optimized for each substrate in the following ranges: AaeUPO
2010 Ultra system, using a fused-silica DB-5HT capillary col-
8
2 2
O
umn (30 m × 0.25 mm internal diameter, 0.1 μm film thick-
ness) from J&W Scientific. The oven was heated from 50 °C
(6–500 nM) with 1.2–10 mM H O MroUPO (0.2–1 μM) with
2 2,
−
1
(
1.5 min) to 90 °C (2 min) at 30 °C min , and then from 90
0
H
.5–10 mM H
, and CglUPO (0.2–10 μM) with 2.5–10 mM H
those resulting in maximal conversion were used). Final
concentrations were selected for maximal transforma-
2 2
O , rCciUPO (0.2–1.2 μM) with 0.5–10 mM
−
1
°
C to 250 °C (15 min) at 8 °C min . The injection was
O
2 2
2 2
O
(and
performed at 250 °C and the transfer line was kept at 300 °C.
Compounds were identified by comparing their mass spectra
and retention times with those of the authentic standards
2 2
H O
tion and reaction selectivity. In control experiments, sub-
strates were treated under the same conditions (including
(
4HS, DHS, trans-stilbene epoxide) and quantified from total-
ion peak areas. Substrate conversion rates and relative molar
abundances of products were calculated with relative re-
sponse factors from chromatographic runs of mixtures of au-
thentic standards. Yields of main products were calculated
using standard curves.
H
2
O
2
) but without enzyme.
After 30–60 min reaction, products of stilbene conversion
were extracted with methyl tert-butyl ether and dried under
1
8
2
N . Enzymatic reactions with O-labeled hydrogen peroxide
1
2
8
(H
O
2
, 90% isotopic content) from Sigma-Aldrich (2% w : v
solution) were performed under the same conditions de-
scribed above. N,O-BisIJtrimethylsilyl)trifluoro-acetamide
Supelco) was used to prepare trimethylsilyl (TMS) derivatives
HPLC analyses
(
HPLC analyses were performed with a Shimadzu LC-2030C
of stilbene reaction products that were analyzed by GC-MS.
Due to the relatively higher water solubility of RSV and
oxyRSV compared with stilbene (which made it difficult to ex-
tract them with apolar solvents), products from Pin and RSV
conversion were analyzed in a different way. After 30 or 60
min, reactions were stopped with 200 μL of 50 mM sodium
azide solution by vigorous shaking and compounds present
were analyzed by HPLC. Definitions of some transformation
parameters are provided in ESI.†
3
D system equipped with a photo diode array detector using
a reversed phase column Agilent InfinityLab Poroshell 120
Bonus-RP C18 (4.6 mm diameter by 150 mm length, 2.7 μm
particle size). The column was isocratically eluted at 40 °C
−
1
and 1 mL min with 0.1% phosphoric acid (pH 2.2) and ace-
tonitrile, 90 : 10, for 2 min, followed by an 18 min linear gra-
dient to 70% acetonitrile, finally held for 3 min. Oxidation
products were identified by comparing their retention times
and spectral data with authentic standards (Pin, RSV and
OxyRSV). Quantification was obtained from peak areas
recorded at 303 nm, and substrate conversion rates and rela-
tive abundances of products were calculated as described
above.
Enzyme kinetics
The kinetics of stilbene dihydroxylation was studied in two
steps. First, stilbene reactions were carried out in 1 mL vials
with 3–200 μM substrate; 32 nM MroUPO; 40 nM rCciUPO; 4
nM AaeUPO; 8 mM ascorbic acid; 20% (v/v) acetone. The sec-
ond hydroxylation step was studied analogously with 3–800
μM of 4-hydroxy-trans-stilbene; 13 nM MroUPO; 120 nM
rCciUPO; 4 nM AaeUPO; 8 mM ascorbic acid; 20% (v/v) ace-
tone. The enzyme concentrations were chosen to obtain simi-
lar transformation degrees in both reaction steps. The reac-
tions were initiated with 0.5 mM H O and stopped after 10 s
UPO molecular structures
Both the A. aegerita and the M. rotula UPOs have been crys-
tallized and their solved molecular structures are available at
the Protein Data Bank (www.rcsb.org/pdb) with ID 2YP1 and
5FUJ, respectively, among other entries. In the present study,
homology models for the C. cinerea and C. globosum UPOs
were obtained at the SWISS-MODEL protein structure
2
2
by vigorous shaking with 200 μL of 50 mM sodium azide.
homology-modelling
server
(https://swissmodel.expasy.
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28,29
org).
PyMOL Molecular Graphics System, ver 2.0
Schrödinger, LLC (https://pymol.org) and Swiss-PdbViewer
ver 4.1 (https://spdbv.vital-it.ch) were used to examine and il-
lustrate the above UPOs' molecular structures.
Results and discussion
Hydroxylation of stilbene by several UPOs
In the present work, several fungal peroxygenases namely
AaeUPO, rCciUPO, MroUPO and CglUPO, were tested for their
ability to hydroxylate trans-stilbene using H O as co-
2
2
substrate (electron acceptor and oxygen source) (Table 1).
Complete conversion (100%) was achieved with all UPOs.
However, very different regioselectivities were accomplished
by the basidiomycetous and ascomycetous UPOs (Fig. 1A).
The former (AaeUPO, rCciUPO and MroUPO) hydroxylated se-
lectively the para-positions of the substrate to form 4,4′-
dihydroxy-trans-stilbene (DHS) (about 99% of total products),
which was identified by comparing GC-MS data (mass spec-
trum, retention time) with an authentic standard (ESI,† Fig.
S3A and S4A-B, respectively). At shorter incubation times the
monohydroxylated 4HS was detected (Fig. 1B). Traces of a tri-
hydroxylated-trans-stilbene (THS), most probably formed by
hydroxylation of DHS, were also found (Table 1). In contrast,
CglUPO did not hydroxylate the aromatic rings and instead
formed exclusively the epoxide (trans-stilbene epoxide) in the
aliphatic chain of trans-stilbene. trans-Stilbene epoxide was
unambiguously identified by GC-MS, comparing its mass
spectrum and retention time with an authentic standard (Fig.
S5A and S4C-D,† respectively). Product yields (of both DHS
and the epoxide) were over 90% for all the UPOs tested. The
yield for DHS is much higher than that of chemical synthesis
Fig.
1
GC-MS analysis of trans-stilbene reactions (60 min) with
AaeUPO, rCciUPO, MroUPO and CglUPO and control (without enzyme)
showing the remaining substrate ( _S _t , trans-stilbene), and the di-
hydroxylated (DHS, 4,4′-dihydroxy-trans-stilbene) and epoxide (St-ep-
oxide; trans-stilbene epoxide) derivatives (A) and GC-MS analysis of
stilbene reactions with AaeUPO at different reaction times (0 min, 1
min, 15 min and 60 min) showing the remaining substrate ( _S _t ), the
monohydroxylated (4HS, 4-hydroxy-trans-stilbene) and dihydroxylated
(DHS) derivatives (B).
CglUPO reaction, a 90% shift from the natural abundance at
m/z 105, m/z 118, m/z 195 and m/z 196 ([M] ) found in the re-
action with unlabeled H O to m/z 107, m/z 120, m/z 197 and
2 2
m/z 198 was ascertained.
+
1
8
(only 25%) (Scheme S1†).
O-labeling studies were
18
performed in trans-stilbene reactions using H2 O2 as UPO
co-substrate to confirm the source of oxygen incorporated
during the formation of both, DHS and the epoxide (Fig. S3B
and S5B†). Mass spectral analysis of the resulting dihydroxy-
lated TMS-derivatives (Fig. S3†) showed that the characteristic
molecular ion fragments had shifted by approx. 90% from
the natural abundance at m/z 356 (found in the reaction with
Kinetics of stilbene oxygenation by UPOs
The estimated kinetic parameters for trans-stilbene hydroxyl-
ation by UPOs are summarized in Table 2. AaeUPO was the
most efficient enzyme catalyzing the double hydroxylation of
trans-stilbene. While the Michaelis–Menten constants (Km)
were similar for all enzymes, the turnover numbers (kcat) were
much higher for AaeUPO resulting in a 15- and 60-fold in-
1
6
ordinary H2 O ; Fig. S3A†) to m/z 360 (Fig. S3B;† 10% of the
2
18
original fragments remained in the H
2 2
O reactions owing to
the 90% isotopic purity of the labeled peroxide). Additionally,
in the mass spectrum of the epoxy derivative from the
crease in the catalytic efficiency (kcat/K ) for the first and
m
Table 1 Conversion of trans-stilbene (0.1 mM) at 60 min by several peroxygenases and relative molar abundance (%) of different products: 4,4′-di-
hydroxystilbene (DHS), trihydroxystilbene (THS) and stilbene epoxide. The molar yield of the main product (DHS or epoxide) with respect to the substrate
is also shown
Products (relative molar %)
Conversion
molar %)
Main product
yield (molar %)
(
DHS
THS
Epoxide
a
c
AaeUPO (12 nM)
100
100
100
100
98.6
98.6
99.1
—
1.4
1.4
0.9
—
—
—
—
100
94 ± 13
91 ± 7
a
b
c
rCciUPO (1.2 μM)
MroUPO (0.2 μM)
c
96 ± 11
b
d
CglUPO (3 μM)
95 ± 3
a
b
c
d
5
2 2
mM H O .
2 2
10 mM H O . DHS. Epoxide.
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Table
3
Catalytic performance of several peroxygenases for
second hydroxylation step, respectively. It was also found that
trans-stilbene hydroxylation
4
HS hydroxylation by AaeUPO was more favorable than that
of trans-stilbene as it exhibited higher kcat and lower K
m
−1
TTN
TOF (s )
values, which increased the catalytic efficiency four times. On
the other hand, catalytic efficiencies were roughly in the
AaeUPO
rCciUPO
MroUPO
200 000
1400
26 000
55.6
0.4
7.2
5
−1 −1
same range (∼10 M s ) for both hydroxylation steps in the
case of MroUPO and rCciUPO. Compared to most other
AaeUPO substrates susceptible to aromatic hydroxylation, the
catalytic efficiency for trans-stilbene turned out to be one to
two orders of magnitude higher (e.g. for the oxygenation of
Substrate conversion and product identification was
followed/achieved by HPLC (Fig. 2) comparing respective data
with authentic standards (Fig. S6†). In Pin reactions, different
conversion rates and product patterns were observed in depen-
dence of the particular UPO. While AaeUPO almost completely
converted Pin (up to 97%) and rCciUPO did it to large extent
(80%), just a low conversion rate (11%) was obtained with
MroUPO and no conversion was observed with CglUPO. The
conversion of Pin by AaeUPO and rCciUPO yielded RSV that
was in turn oxidized to oxyRSV (up to 78% of oxidized product
when the highest AaeUPO dose was used) but, in the case of
rCciUPO, a significant amount of another monohydroxylated
compound tentatively assigned as 2′-hydroxypinosylvin
(oxypinosylvin, oxyPin) was detected (being very minor in the
AaeUPO reactions). This suggest that AaeUPO is more selective
towards the position 4′ of Pin yielding RSV, while similar
amounts of RSV and oxyPin are formed by rCciUPO hydroxyl-
ation of Pin at the 4′ and 2′ positions, respectively. With
MroUPO, RSV was the only product obtained. When using RSV
as substrate, up to 72% and 22% conversion was attained with
AaeUPO and rCciUPO, respectively, but no conversion was ob-
served with CglUPO and MroUPO. The reaction of RSV with the
two former enzymes generated oxyRSV.
5
4
−1 −1
naphtalene and propranolol, 5 × 10 and 4 × 10 M s , re-
spectively, were reported).
3
0,31
This fact can be explained by
the higher Km values observed for these substrates reflecting
lower affinities to the enzyme compared to trans-stilbene.
Similar applies to stilbene hydroxylation by MroUPO, which
gave a catalytic efficiency one order of magnitude higher than
1
4
that reported for naphtalene.
The catalytic differences between the UPOs oxyfunctionalizing
trans-stilbene were supported by their reaction performance. In
the case of AaeUPO, TTN for trans-stilbene hydroxylation (982
μM for DHS and 691 μM for 4HS) raised up to 200 000, while
using MroUPO and rCciUPO, this number was 8- and 143-fold
lower, respectively (Table 3). Thus, AaeUPO displayed a compara-
bly high TTN for the aromatic oxygenation of trans-stilbene as
for the benzylic hydroxylation of alkylbenzenes, for which a TTN
14
of 110 000 was achieved. Very recently, the synthesis of
stilbenoid derivatives by a cytochrome P450 monooxygenase
(
CYP154E1) from Thermobifida fusca and some of its variants
32
has been reported. The enzyme showed a similar para-
hydroxylating regioselectivity for trans-stilbene and similar con-
version rates as reported herein for the UPOs but with consider-
ably lower TTN (4800) as compared with AaeUPO (200 000) and
MroUPO (25 000). Comparing kinetic data, CYP154E1 and its var-
iants exhibited lower affinities (higher Km values) and turnover
numbers, and hence a thousand-times lower catalytic efficiency
compared to AaeUPO.
Table 4 Conversion of 0.1 mM pinosylvin (Pin) and resveratrol (RSV) by
four UPOs yielding RSV, oxyresveratrol (oxyRSV) and oxypinosylvin
2 2
(oxyPin) (different enzyme doses, reaction times and H O concentrations
are compared)
Products
relative molar %)
Hydroxylation of stilbene analogs by several UPOs
(
Conversion
(%)
In addition to DHS synthesis from trans-stilbene, other
stilbenoids (trans-stilbene hydroxylated derivatives) such as
Pin and RSV were also tested as UPO substrates (Table 4).
Enzyme
Substrate
RSV
oxyRSV
oxyPin
AaeUPO
0.025 μM
a
Pin
96.8
94.0
95.9
71.9
62.8
37.3
21.8
—
35.2
62.7
78.2
100
2.0
—
—
—
a
0
0
.050 μM
.100 μM
Pin
b
Pin
a
Table 2 Estimated kinetic parameters for trans-stilbene and 4-hydroxy-
trans-stilbene hydroxylation with several peroxygenases (AaeUPO,
MroUPO and rCciUPO). Data represent mean values of three replicates
with standard errors
0.144 μM
RSV
rCciUPO
c
0
0
.1 μM
.1 μM
Pin
80.4
22.2
39.6
—
27.0
100
33.5
—
b
RSV
−1
−1 −1
kcat (s
)
K
m
(μM)
kcat/K
m
(M
s
)
MroUPO
c
St → 4HS
AaeUPO
rCciUPO
MroUPO
1
1
μM
μM
Pin
10.7
0
100
—
—
—
—
—
6
b
85.7 ± 8.5
9.1 ± 0.8
4.2 ± 0.2
20.8 ± 5.6
30.5 ± 7.0
15.3 ± 2.5
4.1 ± 1.2 × 10
3.0 ± 0.7 × 10
2.7 ± 0.5 × 10
RSV
5
5
CglUPO
a
1
1
0 μM
0 μM
Pin
0
0
—
—
—
—
—
—
b
4HS → DHS
RSV
7
5
5
AaeUPO
rCciUPO
MroUPO
225 ± 48.3
6.6 ± 1.3
11.7 ± 0.3
14.1 ± 7.7
18.9 ± 8.5
46.4 ± 2.3
1.6 ± 0.9 × 10
3.5 ± 0.8 × 10
2.5 ± 0.1 × 10
a
b
30 min reactions with 2.5 mM H
2 2
O .
60 min reactions with 5 mM
c
H
2 2 2 2
O . 30 min reactions with 5 mM H O .
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Fig. 3 Solvent access surface in AaeUPO (A), MroUPO (B), rCciUPO (C)
and CglUPO (D) showing different: i) sizes of the channel giving access
to the heme cofactor (sticks); and ii) phenylalanine residues (magenta
surface) at the channel edge and other surface regions. Cenital views
showing the channels giving access to the distal side of the cofactor
pocket on the heme iron atom (one acetate molecule is shown inside
the MroUPO channel). From 2YP1 (A), 5FUJ (C) and two homology
models (B and D). Residue numbering does not includes the signal
peptide.
Fig. 2 HPLC analysis of pinosylvin (Pin, left) and resveratrol (RSV,
right) reactions with AaeUPO, rCciUPO, MroUPO and CglUPO showing
the remaining substrates ( P_ _i n_ , R_ _S V_ ) and the products, RSV,
oxyresveratrol (oxyRSV), and the trihydroxylated derivative of Pin
(
oxyPin).
4
+
ferryl oxygen (Fe O) of activated heme (UPO compound I).
The same would apply to other stilbenoids investigated in the
present study. Less ‘perfect’ positioning of the substrate in
the more aliphatic environment of rCciUPO and MroUPO
channels would result in lower hydroxylating efficiency both
for stilbene (14–15 times lower) and 4HS (45–65 times lower)
(Table 2). For MroUPO, the catalytic efficiencies were similar
to those reported for other UPO substrates like veratryl alco-
Molecular architecture vs. UPO activity
The above differences in the regioselectivity and kinetic con-
stants for stilbene oxidation (Tables 1 and 2) should be relat-
able to differences in the UPOs' molecular structure affecting
the specific residues putatively involved in catalysis. These
would be located at: i) the access channel leading to the bur-
ied heme cofactor and its surface opening; and ii) the enzyme
active site itself, i.e. the heme pocket that follows the access
channel.
1
4
hol or benzyl alcohol, but the catalytic efficiencies of
AaeUPO for the oxyfunctionalization of stilbene and especially
of 4HS turned out to be much better than the values reported
Besides the obvious difference in molecular size – ‘long’
AaeUPO and rCciUPO (Fig. 3 left) consist of 325 and 337
amino acids (mature protein), respectively, while ‘short’
9
for any other ‘two-electron oxidation’ substrate. The oxygena-
tion results also show that UPOs hydroxylate the phenyl moi-
ety of stilbenoids with preference respect to the 4-OH–phenyl
moiety. This is revealed by the production of DHS in the stil-
bene reaction. The complete reaction most probably involves
4HS release from the enzyme and re-entering with the second
aromatic ring pointing towards the heme cofactor.
The regioselectivity of CglUPO in stilbene oxygenation rad-
ically differed from that of aromatic oxygenation discussed
above, since ethenyl epoxidation was produced. Interestingly,
the UPO of the ascomycete C. globosum (A. aegerita, M. rotula
and C. cinerea are basidiomycetes), has a noticeably different
active site. Of the five residues above the heme plane (Fig. 4),
MroUPO and CglUPO (Fig. 3 right) only comprise 236 and
8
2
42 residues, respectively – the most pronounced difference
is the distribution of solvent-exposed phenylalanines.
AaeUPO possesses four phenylalanine residues (Phe69,
Phe191, Phe199 and Phe274) at the entrance of the heme ac-
cess channel, which make it rather hydrophobic and affine to
aromatic rings, while MroUPO and rCciUPO only have one
(Fig. 3). It is conceivable that aromatic interactions between
the substrate rings and these four phenylalanine residues
may contribute to optimal positioning of stilbene (and later
of 4HS) with the para-position at reaction distance of the
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in contrast to other UPOs, CglUPO also specifically epoxi-
1
8
dized testosterone – will have to be clarified in future muta-
genesis studies. Although a yeast expression system for
AaeUPO has been developed after enzyme directed evolu-
3
3
tion, new procedures and hosts for expressing wild-type
genes of this and other UPOs are required for investigating
active-site residues and engineering UPO biocatalysts for se-
3
4
lective oxygenation reactions of biotechnological interest.
Conclusions
We report here on a new enzymatic route for the production
of DHS from trans-stilbene and RSV from Pin (Scheme 1) in
one-step by fungal peroxygenases (UPOs). The potent effect of
DHS in inhibition of cancer invasion and metastasis has re-
6
cently been reported “in vivo”. Usually, DHS is obtained by
chemical synthesis, which involves several-steps, the use of
metal catalysts, etc. (Scheme S1†). The enzymatic reaction
with UPOs represents a smart and environmentally sound al-
ternative to chemical synthesis. In addition, the UPO-
catalyzed synthesis of the potent antioxidants and free radical
scavengers RSV (and oxyRSV) from Pin (a natural constituent
of pine wood) is also reported here for the first time. A pre-
liminary analysis of the active site cavities and access chan-
nels has revealed differences that may explain and affect the
reactivity and regioselectivity of the different UPOs in the stil-
bene oxygenation, but more structural and functional infor-
mation is required for reaction understanding and enzyme
engineering.
Fig. 4 Active site residues, including those putatively involved in
catalysis, in AaeUPO (A), and homologous residues in MroUPO (B),
rCciUPO (C) and CglUPO (D): five residues above the cofactor (heme
pocket “distal” side) plus the cysteine acting as ligand of the heme iron
below the cofactor (pocket “proximal” side) are shown, together with
an acetate molecule (Ac) at the substrate position in AaeUPO and
MroUPO. Black labels correspond to residues as found in AaeUPO,
while varying residues have color labels (blue, red or green). See Fig. 3
for model references.
it only shares with AaeUPO the glutamic residue (CglUPO
Glu158) putativelly involved in the binding and fission of
H O (ref. 8) (also conserved in the other two UPOs). An adja-
2 2
cent residue is also thought to contribute to this reaction (as
charge stabilizer), being either an arginine (in AaeUPO and
CciUPO; Arg189) or a histidine (in MroUPO and CglUPO;
His86 and His88, respectively). CglUPO also shares with
MroUPO the absence of a phenylalanine near the heme edge
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
(homologous to AaeUPO Phe121). However, the main pecu-
liarities of CglUPO's active site are: i) the absence of a con-
served phenylalanine (homologous to AaeUPO Phe199, al-
This study was funded by the EnzOx2 (H2020-BBI-PPP-2015-
2
5
-1-720297) EU-project, and the BIORENZYMERY (AGL2014-
3730-R) project of the Spanish MINECO (co-financed by
though Phe154 occupies
a neighbor position) and in
particular, ii) the presence of a unique and potentially reac-
tive tyrosine (Tyr162) in the middle of the active site.
Whether this tyrosine is responsible for the preferred epoxi-
dation of isolated double bonds within complex molecules –
FEDER). H. Lund (Novozymes) is acknowledged for
rCciUPO.
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