Selective synthesis of 4-hydroxyisophorone and 4-ketoisophorone by fungal peroxygenases

Article abstract of DOI:10.1039/c8cy02114g

The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosum and Humicola insolens were capable of selectively hydroxylating isophorone to 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegerita was less regioselective, forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolens UPO full kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotula and Coprinopsis cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE software, and the distances from heme-bound oxygen in H₂O₂-activated enzyme to different substrate atoms, and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion of 10 mM isophorone was achieved with H. insolens UPO within nine hours, with total turnover numbers up to 5500. These biocatalysts, which only require H₂O₂ for activation, may represent a novel, simple and environmentally-friendly route for the production of isophorone derivatives.

Full text of DOI:10.1039/c8cy02114g

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Selective synthesis of 4-hydroxyisophorone and
4-ketoisophorone by fungal peroxygenases†
Cite this: DOI: 10.1039/c8cy02114g
a
b
bc
d
d
Carmen Aranda, Martí Municoy, Víctor Guallar, Jan Kiebist, Katrin Scheibner,
e
a
e
René Ullrich, José C. del Río, Martin Hofrichter,
Angel T. Martínez * and Ana Gutiérrez
f
a
*
The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosum
and Humicola insolens were capable of selectively hydroxylating isophorone to 4-hydroxyisophorone
(4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-
and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegerita was less regioselective,
forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most
stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolens UPO full
kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the
R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotula and Coprinopsis
cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational
simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE soft-
ware, and the distances from heme-bound oxygen in H O -activated enzyme to different substrate atoms,
Received 11th October 2018,
Accepted 7th December 2018
2
2
and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion
of 10 mM isophorone was achieved with H. insolens UPO within nine hours, with total turnover numbers
DOI: 10.1039/c8cy02114g
2 2
up to 5500. These biocatalysts, which only require H O for activation, may represent a novel, simple and
rsc.li/catalysis
environmentally-friendly route for the production of isophorone derivatives.
be the solution, using copperIJII) acetylacetone or
molybdenum-based systems as catalysts, but required toxic
Introduction
5
Isophorone derivatives, such as 4-hydroxyisophorone (4HIP)
and 4-ketoisophorone (4KIP), are of interest as flavour-and-
heavy metals and led to the formation of undesired side prod-
ucts. Moreover, a direct chemical oxidation process of iso-
phorone to 4HIP is not available and this compound is usu-
ally synthesized by reduction of 4KIP, which can be a rather
expensive starting material.
1
fragrance additives, and as intermediates in the synthesis of
2
,3
6,7
pharmaceuticals, vitamins and natural pigments. A variety
of chemical methods is available for the production of 4HIP
and 4KIP. Thus, both derivatives have been synthesized from
Alternatively, some biological processes for the synthesis
of 4HIP and 4KIP have been described, often using cyto-
chrome P450 monooxygenases (P450s). Among them, the
microbial biotransformation of isophorone was described for
fungi – like Aspergillus niger, Alternaria alternata and Neuros-
1
,4
β-isophorone,
which – on its part – can be obtained by
isomerization of isophorone (also known as α-isophorone).
The rearrangement of β-isophorone to the α-isomer, however,
is a main drawback of this process. The direct oxidation of
8
,9
2
isophorone to 4KIP with molecular oxygen (O ) appeared to
pora crassa – with 4HIP and 7-hydroxyisophorone (7HIP) as
main metabolites. More recently, a process using recombi-
nant Escherichia coli transformed with the P450-BM3 gene (to-
gether with the gene of NADPH-regenerating glucose dehydro-
genase) allowed the scaled-up selective production of 4HIP at
a
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes
0, E-41012 Seville, Spain. E-mail: anagu@irnase.csic.es
1
b
Barcelona Supercomputing Center, Jordi Girona 31, E-08034, Barcelona, Spain
1
0
c
kilogram scale. On the other hand, 4KIP has been produced
either in an one-pot two-step enzymatic process or as a cas-
cade process employing cells co-expressing P450-WAL and
ICREA Passeig Lluís Companys 23, E-08010, Barcelona, Spain
JenaBios GmbH, Löbstedter Str. 80, 07749 Jena, Germany
d
e
TU Dresden, Department of Bio- and Environmental Sciences, Markt 23, 02763
Zittau, Germany
11
Cm-ADH10 dehydrogenase. However, isolated P450s gener-
f
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040
ally suffer the disadvantage of rather higher instability and
the frequent need of auxiliary enzymes/domains and expen-
sive cofactors.
Madrid, Spain. E-mail: atmartinez@cib.csic.es
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8cy02114g
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Catalysis Science & Technology
Unspecific peroxygenases (UPOs, EC.1.11.2.1) are novel
and appealing biocatalysts for organic synthesis, since their
and UV-visible spectroscopy following the characteristic
heme-maximum around 420 nm (Soret band of resting-state
heme-thiolate proteins). Enzyme concentration was estimated
according to the characteristic UV-vis band of the reduced
2 2
‘simplicity’ (only H O is required for activation) and stability
(as secreted enzymes) circumvent major disadvantages of
3
3
P450s while catalyzing the same kind of oxyfunctionalization
UPO-complex with carbon monoxide.
1
2
reactions. The first enzyme of this class was discovered in
1
3
2
004 in the basidiomycete Agrocybe aegerita and since then,
1
4
Model compounds
new peroxygenases came out from Coprinellus radians,
Marasmius rotula, and more recently from Chaetomium
1
5
3,5,5-Trimethyl-2-cyclohexen-1-one (isophorone) from Sigma
Aldrich (97% purity) was tested as substrate of the
above UPOs. 3,5,5-Trimethyl-2-cyclohexen-1,4-dione (4-keto-
isophorone, 4KIP) also from Sigma Aldrich, and chemically-
1
6
globosum. Their widespread occurrence in the fungal king-
dom has been demonstrated by the analysis of basidiomy-
cete, ascomycete and other fungal genomes and revealed over
one-thousand putative peroxygenase genes. This allowed
the production of recombinant enzymes, like those of
1
7
synthesized
4-hydroxy-3,5,5-trimethyl-2-cyclohexen-1-one
(4-hydroxyisophorone, 4HIP) by 4KIP reduction, were used as
standards in gas chromatography-mass spectrometry (GC-MS)
analyses. 4HIP, obtained as a racemic mixture by chemical re-
1
8
16
Coprinopsis cinerea
or Humicola insolens,
which are
heterologously expressed by Novozymes A/S (Bagsvaerd, Den-
mark) in the mold Aspergillus oryzae.
7
duction of 4KIP, was used as substrate in enzymatic reac-
The spectrum of reactions catalyzed by these enzymes is
steadily increasing and includes oxygenations of both aro-
tions together with isophorone.
1
9,20
21–24
matic
and aliphatic compounds,
fatty acids epoxida-
2
5
26
Enzyme reactions
tion and chain-shortening, and also reactions of rather
complex and bulky substrates like steroids,
osteroids
2
7,28
and sec-
Reactions (1 mL volume) with isophorone (0.1 mM) were
performed at 30 °C, in 50 mM phosphate buffer, pH 7. The
enzyme concentrations ranged from 50 nM to 10 μM, using
2.5 to 5 mM H O . In control experiments, substrates were
2
9,30
that are subject to epoxidation, side-chain hy-
droxylation or side-chain removal.
In the present work, the hydroxylation of isophorone by
several UPOs with different selectivities is presented for the
first time, to be included in the portfolio of reactions cata-
2
2
treated under the same conditions (including H O ) but with-
2
2
out enzyme. After 30 min reaction, products were extracted
with methyl tert-butyl ether, which was evaporated under N2,
and the products dissolved in chloroform to be analyzed by
GC-MS. Reactions at higher isophorone concentration (10
mM) were performed with CglUPO and rHinUPO (2–5 μM).
H O was added with a syringe pump to give concentrations
1
2,17
lyzed by these novel and exciting enzymes.
Materials and methods
Enzymes
2
2
−
1
AaeUPO (isoform II, 46 kDa), the first UPO described in 2004,
is a wild-type (i.e. non-recombinant) peroxygenase from cul-
tures of A. aegerita TM-A1, grown in soybean-peptone me-
dium, which was purified as described by Ullrich and
in the reaction mixture of 1 or 5 mM h during 48 h or 12 h,
respectively. Reactions for chiral analyses were carried out
with 10 mM isophorone or 4HIP (racemic mixture) and 5 μM
enzyme (CglUPO, rHinUPO and AaeUPO) for 60 min, with
H O manually added in small doses to a final concentration
3
1
Hofrichter. MroUPO is another wild-type peroxygenase (32
kDa) from cultures of M. rotula DSM-25031 (German Collec-
tion of Microorganisms and Cell Cultures, Braunschweig),
2
2
of 5–20 mM. Products were extracted with ethyl acetate and
directly analyzed by GC-MS or dried and dissolved in the mo-
bile phase to be analyzed by HPLC.
1
5
which was purified as described by Gröbe et al. CglUPO (36
kDa) is a third wild-type peroxygenase from cultures of C.
globosum DSM-62110, which was purified as recently de-
1
6
Enzyme kinetics
scribed by Kiebist et al. The recombinant enzymes rCciUPO
44 kDa) and rHinUPO were provided by Novozymes A/S.
(
Reactions were carried out with 6.25–6400 μM substrate and
rCciUPO corresponds to the protein model 7249 from the se-
quenced C. cinerea genome available at the JGI (http://
2 2
100 nM enzyme. They were initiated adding 0.5 mM H O
and stopped by vigorous shaking in 5 mM sodium azide. Re-
action times, the reaction velocity of which was linear, were
previously selected: 5 min for AaeUPO, 3 min for CglUPO and
1 min for rHinUPO. All reactions were performed in tripli-
cates. Product quantification was carried out by GC-MS using
external standard curves, and kinetic parameters – turnover
2
2,27,30
genome.jgi.doe.gov/Copci1) used in several studies.
The rHinUPO sequence has been more recently reported
and used for oxyfunctionalizations that are not catalyzed by
3
2
1
6
other UPOs. Both UPOs were expressed by Novozymes in As-
pergillus oryzae (patent WO/2008/119780). All UPO proteins
were purified by fast protein liquid chromatography (FPLC)
using a combination of size exclusion chromatography (SEC)
and ion exchange chromatography on different anion and
cation exchangers. Purification was confirmed by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
number (kcat), Michaelis constant (K ) and catalytic efficiency
m
(kcat/K ) – were obtained by fitting the data to the Michaelis–
m
Menten equation, or to the corresponding variation of this
equation when substrate inhibition is occurring, using
SigmaPlot (Systat Software Inc., San Jose, CA, USA).
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GC-MS
gorithm performs: 1) ligand perturbation (translation and ro-
tation); 2) protein perturbation following normal modes; 3)
explicit water sampling; 4) side chain prediction; and 5) over-
all minimization. The final structure is then accepted or
rejected based on a Metropolis criterion. The adaptive proto-
col improves PELE's sampling by running multiple short sim-
ulations (epochs) where initial conditions are selected
through a reward function aiming at sampling non-visited
areas. We also used a new version of PELE that allows for ex-
plicit water sampling, where water molecules are allowed to
freely move (with 100 small translations and rotations) after
the backbone sampling. Two sets of simulations were
performed for each ligand and structure. In the initial one the
ligand was placed on the protein surface, next to the entrance
to the active site (at ∼16 Å from the heme's iron atom), and
allowed to diffuse freely into the heme distal site. The struc-
ture with the best (ligand) binding energy from the initial
simulation was then selected for a second local refinement
run, where the ligand center of mass was constrained to move
within 8 Å from the heme's iron. All simulations used 50
epochs of 16 MC PELE steps each with 128 computing cores.
The analyses were performed in a Shimadzu GC-MS QP 2010
Ultra system, using a fused-silica DB-5HT capillary column
(30 m × 0.25 mm internal diameter, 0.1 μm film thickness)
from J&W Scientific. The oven was heated from 50 °C (1.5
min) to 90 °C (2 min) at 30 °C min , and then from 90 °C to
−
1
−
1
2
50 °C (15 min) at 8 °C min . The injection was performed
at 250 °C and the transfer line was kept at 300 °C. Com-
pounds were identified by comparing their mass spectra and
retention times with those of available commercial or synthe-
sized authentic standards, and by search in the NIST library.
Chiral HPLC
Chiral analyses were performed with a Shimadzu LC-2030C
3
D system equipped with a photo-diode array detector using
a chiral column Chiralpak IG (5 μM particle size, 4.6 mm di-
ameter × 150 mm, Daicel Chemical Industries Ltd.) equipped
with a Chiralpak IG guard column (5 μM particle size, 4.0
mm diameter × 10 mm). The column was eluted in isocratic
mode with 95% hexane and 5% isopropanol at 0.5 mL min
for 60 min, and the absorbance was monitored at 254 nm.
Enantiomers were identified based on the elution order previ-
−
1
−
1
Interaction/binding energies (kcal mol ) were derived as Eab
− (Ea + Eb), where Eab is the total energy of the complex, Eb
the energy of the ligand and Ea the energy of receptor (every-
thing but the ligand), all of them obtained at the OPLS2005
level of theory with a surface GB implicit solvent model.
3
4
ously reported.
Molecular models
AaeUPO and MroUPO models were obtained from the chain-A
of the 2YP1 and 5FUJ crystal structures, after removing the
Cys227 dimerization disulfide bridge from the second struc-
ture. Homology models for CglUPO, rHinUPO and rCciUPO
Results and discussion
Regioselectivity in isophorone transformation by UPOs
In the present work, the ability of several UPOs to oxidize iso-
phorone was analyzed, and different transformation patterns
were observed (Fig. 1). The peroxygenases from the ascomy-
cetes C. globosum (CglUPO) and H. insolens (rHinUPO), and
the basidiomycete A. aegerita (AaeUPO) were found to trans-
form the substrate, although with different regioselectivities
3
5,36
were obtained at the Swiss-Model server,
with related
crystal structures as templates. The heme cofactor and the
Mg ion (along with its two coordinated water molecules) were
then superimposed using 2YP1 and 5FUJ as templates,
followed by an initial minimization to release steric clashes.
An oxygen atom (iron-oxo) was finally added to all structures
modeling heme compound I. All systems were prepared at
(
Table 1). In contrast, the enzymes from the basidiomycetes
C. cinerea (rCciUPO) and M. rotula (MroUPO) were unable to
3
7
pH 7 with the protein preparation wizard from Schrödinger.
Heme charges were obtained from a quantum mechanics/
molecular mechanics (QM/MM) minimization using QSite at
the DFT M06-LIJlacvp*)/OPLS level of theory. Based on previ-
3
8
ous experience in heme-bound systems, the charge of the
Mg ion was set to 1.2. isophorone and 4HIP (S- and
R-enantiomers) substrates were built with Maestro and opti-
mized at the OPLS level of theory. In addition, two explicit
water molecules were placed in the active site when exploring
diffusion of the ligands. The presence of a water molecule
has been highlighted in compound I activity and might be
3
9
important when diffusing polar substrates such as 4HIP.
Ligand diffusion simulations
4
0
The new adaptive-PELE software was used to study ligand
diffusion and binding on the different UPO structures. PELE
uses a Monte Carlo (MC) procedure to describe the protein-
ligand conformational dynamics. At each MC iteration, the al-
Fig. 1 GC-MS analysis of 0.1 mM isophorone reactions (30 min) with
0
.1 μM of MroUPO, rCciUPO, CglUPO, rHinUPO, AaeUPO and control
without enzyme, showing the remaining substrate (IP, isophorone) and
the hydroxylated (4HIP and 7HIP) and oxo (4KIP and 7FIP) derivatives.
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Table 1 Comparison of five UPOs in isophorone (0.1 mM) conversion (%
of substrate) and relative abundance of the products 4HIP, 7HIP, 4KIP
and 7FIP (% of the total products) after 30 min reaction, using different
enzyme and peroxide doses
4
HIP to 4KIP of 1 : 1 (compared to 4 : 1 in the case of CglUPO)
(
Table 1).
AaeUPO was found to be less regioselective in oxidizing
isophorone, since – in addition to 4HIP – other mono-
hydroxylated and keto-derivatives were formed (Scheme 1B).
These side products were identified as 7HIP and
7-formylisophorone (7FIP) due to their mass spectra (Fig. S3†)
that matched with those previously reported, with the sin-
gular mass fragment at m/z 125 in 7HIP, different from that
at m/z 112 in 4HIP.
Conversion
4HIP
7HIP
4KIP
7FIP
a,d
b,d
AaeUPO
AaeUPO
CglUPO
CglUPO
72
96
95
100
84
100
46
46
79
—
53
3
25
19
—
—
—
—
tr
tr
20
100
45
97
29
35
1
—
2
a,d
c,e
34
a,d
c,e
rHinUPO
rHinUPO
—
Reactions with low AaeUPO dose (0.1 μM) showed that hy-
droxylation took place in similar proportion at C (46%) and
4
C7 (54%), but 7HIP was rapidly over-oxidized to form the cor-
responding aldehyde, and 7FIP and 4HIP were the major fi-
nal products of the reaction (Table 1). Interestingly, 4HIP was
barely further oxidized to 4KIP.
a
b
Reactions using 0.1 μM UPO. d Reactions using 0.25 μM UPO.
c
e
Reactions using 0.5 μM UPO. Reactions using 2.5 mM H
Reactions using 5 mM H ; tr denotes traces.
2 2
O .
2
O
2
convert the substrate, even at the highest (10 μM) enzyme
doses tested.
The regioselectivity observed in the hydroxylation of iso-
phorone by some UPOs is similar to that reported for certain
P450s. Among them, P450cam-RhFRed variants have been
reported to yield 4HIP, 7HIP and isophorone oxide (2,3-epoxy-
3,5,5-trimethyl-1-cyclohexanone) as major products, with 4HIP
CglUPO and rHinUPO selectively oxidized isophorone in
position 4 (Scheme 1A) yielding mono-hydroxylated 4HIP and
the keto-derivative 4KIP, which were identified by their reten-
tion times compared with authentic standards (Fig. S1†) and
their mass spectra (Fig. S2†). Besides the 4HIP and 4KIP mo-
lecular ions at m/z 154 and 152, respectively, their mass spec-
tra showed a shift from α-isophorone fragments at m/z 54
and 82 to m/z 70 and 98 in 4HIP and to m/z 68 and 96 in
1
1
as the only product from one of the variants. 4HIP was also
the main product of reactions with CYP102A1 and CYP101A1,
4
KIP, which corresponds to the insertion of a hydroxyl or
keto group. Isophorone conversion by rHinUPO and CglUPO
84 and 95%, respectively) was already efficient at low enzyme
(
dose (0.1 μM) and both enzymes showed the tendency to
over-oxidize 4HIP to form 4KIP. Over-oxidation was more pro-
nounced in the case of rHinUPO as shown by the ratio of
Scheme 1 Isophorone (IP) hydroxylation catalyzed by CglUPO and
rHinUPO (A) and AaeUPO (B), showing the hydroxylated 4HIP
Fig. 2 Time course of 10 mM isophorone (IP) reaction with 5 μM
2 2
CglUPO (A) and rHinUPO (B) and H O (added with a syringe pump to
−1
(
4-hydroxyisophorone) and 7HIP (7-hydroxyisophorone) and the oxo
KIP (4-ketoisophorone) and 7FIP (7-formylisophorone) derivatives.
give 5 mM h concentration), showing substrate and products (4HIP
and 4KIP) concentrations.
4
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Table 2 Kinetic parameters of AaeUPO, CglUPO and rHinUPO for the
Table 4 Results from chiral HPLC analysis of the 10 mM isophorone re-
action (60 min) with AaeUPO, CglUPO and rHinUPO (5 μM) showing the
yields of the R and S enantiomers of 4HIP, and the resulting enantiomeric
excess (ee), together with the amount of 4KIP formed and the total con-
version yield (% of substrate) under the given conditionsa
a
hydroxylation of isophorone
−1
−1 −1
kcat (s
)
K
m
(μM)
kcat/K
m
(mM
s )
AaeUPO
CglUPO
rHinUPO
20.1 ± 1.1
4.4 ± 0.4
42.0 ± 9.8
1380 ± 200
309 ± 55
633 ± 201
14.6 ± 2.2
14.2 ± 2.8
66.4 ± 26.1
Conversion 4HIP
4KIP
R-4HIP S-4HIP ee
(%)
(mM)
(mM)
(%)
(%)
(%)
a
Data represent mean values of three replicates with standard
deviations.
a
AaeUPOa 51
2.5
3.3
2.0
0.1
0.3
0.4
6
30
48
94
70
52
88
40
4
CglUPO 24
rHinUPO 36
b
a
b
Reactions using 20 mM H
2 2
O .
2 2
Reactions using 5 mM H O .
although minor amounts of the epoxide, 7HIP and further ox-
3
4
idation products were observed. P450s, unlike rHinUPO or
CglUPO, seem to be unable to oxidize 4HIP into 4KIP and,
therefore, two enzymes (a P450 and an alcohol dehydroge-
the Km of CglUPO and rHinUPO for isophorone hydroxylation
is similar to values (380–440 μM) reported for CYP102A1 vari-
ants when decoy molecules were used, and the turnover num-
1
1
nase) are necessary to obtain 4KIP from isophorone.
−
1
In view of the higher selectivity to form the products of
interest (4HIP and 4KIP), CglUPO and rHinUPO were selected
to perform reactions with higher (×100) substrate load
bers of these variants (2.5–5.5 s ) were similar as well to that
−
1 34
reported herein for CglUPO (4.4 s ).
Higher total turnover numbers (TTNs) were attained with
rHinUPO (2660) than with CglUPO (1820) under the same re-
action conditions, although these values could be up to 5600
and 3600 when lower enzyme doses were used (Table 3). The
TTNs are in the range of those reported for two-step (1567) or
one-step (3421) isophorone oxidation by the combination of
(Fig. 2). These experiments revealed a faster substrate conver-
sion by rHinUPO that completely transformed isophorone
within 6 h, while CglUPO needed 12 h for 87% conversion. As
expected, a higher proportion of 4KIP was observed in the
rHinUPO reactions. A higher enzyme dose would be needed
to complete conversion into 4KIP, as it was already found
when lower substrate concentrations were tested (Table 1).
1
1
P450-WAL and Cm-ADH10.
UPO enantioselectivity on isophorone and racemic 4HIP
Kinetics of isophorone hydroxylation by UPOs
Enantioselectivity in the synthesis of 4HIP by the three UPOs
hydroxylating isophorone was determined by HPLC (Fig.
S5†). The results of the chiral analysis showed that only the
reaction with AaeUPO can be considered as stereoselective,
with an enantiomeric excess (ee) of 88% S-4HIP (Table 4).
The three UPOs preferentially formed the S-enantiomer, in
Despite the difficulties to determine initial enzymatic reac-
tion rates by GC-MS, kinetic curves for isophorone hydroxyl-
ation by the three UPOs could be obtained (Fig. S4†) and re-
action constants (kcat, K
m
m
and kcat/K ) were estimated
(Table 2). There were differences in enzyme affinities, since
1
1,42
the K
m
values were four- and two-fold higher for AaeUPO
contrast to P450s that rather formed the R-enantiomer.
than for CglUPO and rHinUPO, revealing the higher iso-
phorone affinity of the two latter enzymes. Moreover,
The above values were estimated under reaction condi-
tions where the product concentration was similar for all
rHinUPO displayed a ten-fold higher turnover number (kcat
)
compared to CglUPO, which resulted in five-fold higher cata-
lytic efficiency, while the efficiencies of AaeUPO and CglUPO
were similar.
The catalytic efficiency of these UPOs hydroxylating iso-
phorone is in the range of previously reported for the hydrox-
4
1
ylation of cyclohexane by other UPOs. On the other hand,
Table 3 Product concentration and catalytic performance – given by to-
tal turnover number (TTN) and turnover frequency (TOF) – of CglUPO
and rHinUPO after 9 h reaction using a higher isophorone concentration
(10 mM)
−
1
4
HIP (mM)
4KIP (mM)
TTN
TOF (min )
b
CglUPOa
CglUPO
6.7
4.8
2.3
2.4
1.2
1.2
5.5
4.4
1820
3600
2660
5600
3.4
6.7
4.9
b
Fig. 3 Example of the initial (black) and refinement (red) simulations
rHinUPOa
on isophorone (IP) diffusion (C -oxo distance being monitored with
4
rHinUPO
10.4
respect to binding/interaction energy) on CglUPO, using two-step
adaptive PELE.
a
b
35
Reactions using 2 μM enzyme. Reactions using 5 μM enzyme.
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35
Fig. 4 Isophorone (IP, left) and (S)-4-hydroxyisophorone (S-HIP, right) diffusion refinement on five UPOs, with adaptive PELE monitoring the dis-
tance between the substrate and the oxo atom (red sphere) of the H -activated heme with respect to the binding energy (E ). A) C distance vs.
2
O
2
b
4
energy plot for IP diffusion in CglUPO (red), MroUPO (magenta), AaeUPO (green), rHinUPO (cyan) and rCciUPO (blue) (see Fig. S7† for individual
PELE plots of the five UPO systems). B and C) IP at the two lowest binding-energy positions during the CglUPO and AaeUPO simulations (A), re-
4
spectively. D) H distance vs. energy plot for S-HIP diffusion on CglUPO (red) and AaeUPO (green). E and F) S-HIP at the two lowest binding-energy
positions during the CglUPO and AaeUPO simulations (D), respectively.
three enzymes (2.0–3.3 mM) and 4HIP over-oxidation was
minimal, since it was observed that the ee of the hydroxyl-
ation product changed when 4KIP was formed. This was due
to higher velocity in the conversion of the S-enantiomer com-
pared to the R-enantiomer, as observed in rHinUPO reactions
with racemic 4HIP as substrate (Fig. S6†). That way, a kinetic
resolution of the racemate, with ee of 99–100% and 60–75%
recovery, can be achieved with rHinUPO and CglUPO. In con-
trast, AaeUPO just slowly converted the racemic mixture of
4HIP, as it was also observed in the reaction of the enzyme
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with isophorone, where only traces of 4KIP were formed
shown in Fig. 4B and C). The above results explain the lack
(Fig. 1), and no enantiomeric enrichment was produced.
of C hydroxylation by CglUPO and the similar percentages of
7
7 4
C -derivatives (7HIP + 7FIP) and C -derivatives (4HIP + 4KIP)
by AaeUPO (Table 1).
Computational analyses: molecular modeling
Isophorone diffusion was done in two simulation steps using Conclusions
4
0
PELE software. For the initial simulation, after preparing
all systems placing isophorone on the surface next to the en-
trance channel, the substrate was allowed to move freely
within 16 Å of the heme iron. In all systems, the substrate
moved easily into the active site. Structures in direct contact
with the heme produced the best binding-energy poses, al-
though they presented different substrate orientations. There-
fore, after selecting the best binding-energy structure, we run
a local refinement, where the ligand was forced to move
within ∼8 Å of the heme iron. Fig. 3 shows an example of
these two runs for CglUPO, where we display the binding-
We report a new enzymatic route for isophorone oxidation to
form 4HIP and 4KIP, which are interesting products for the
flavour-and-fragrance and pharmaceutical industries. The di-
rect enzymatic oxidation of isophorone to 4KIP (with only
one enzyme) is reported here for the first time for two fun-
gal peroxygenases (CglUPO and rHinUPO). The above repre-
sents an advantage over the route with P450s, since the latter
needs two enzymes (a P450 and an alcohol dehydrogenase) to
obtain 4KIP from isophorone. However, process optimization
of isophorone conversion by UPO is needed to attain the
high-scale transformations reported for whole-cell P450
4
energy profile with respect to the C -oxo distance for the ini-
tial (black) and the refinement (red) simulations.
4
3
systems.
Fig. 4A shows the binding energy along the C -oxo dis-
4
Conflicts of interest
tance for isophorone in the refinement runs for the five UPO
structures (identified with different colors). Among them,
three UPOs show distances lower than 3 Å: CglUPO (red),
AaeUPO (green) and rHinUPO (cyan) (see Fig. S7† for the indi-
vidual PELE plots). Such positioning will largely facilitate the
hydrogen-atom abstraction by compound I, in agreement
with the experimental results (Table 1). Interestingly, differ-
ent isophorone reactive poses were detected for the different
UPOs, as shown in Fig. 4B and C for CglUPO and AaeUPO, re-
spectively. A closer examination of these structures showed
shorter distances to the heme oxo for the pro-S (1.9 Å in
both) than for the pro-R (3.5 and 2.6 Å, respectively, not
shown) hydrogen atoms, which explained the preferential for-
mation of the 4HIP S-enantiomer, as shown in Table 4.
There are no conflicts to declare.
Acknowledgements
This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-
2-1-720297)
EU-project,
the
AGL2014-53730-R
(BIO-
RENZYMERY) and CTQ2016-79138-R projects of the Spanish
MINECO (co-financed by FEDER) and the CSIC (201740E071)
project. Novozymes (Bagsvaerd, Denmark) is acknowledged
for providing samples of rCciUPO and rHinUPO.
References
The resulting S-4HIP was also diffused with PELE, and
strong differences in the C -oxo distances and energies were
4
1 S. Krill, K. Huthmacher and S. Perrin, US Pat., 6469215B1,
2002.
obtained, as illustrated in Fig. 4D for two of the systems, with
CglUPO closely approaching the heme oxo (Fig. 4E) while no
catalytic distances were attained by AaeUPO (Fig. 4F). The
above results agree with isophorone oxidation to 4KIP by
CglUPO, while 4HIP is the main product from AaeUPO
2 M. Eggersdorfer, D. Laudert, U. Létinois, T. McClymont, J.
Medlock, T. Netscher and W. Bonrath, Angew. Chem., Int.
Ed., 2012, 51, 12960–12990.
3 O. Isler, H. Lindlar, M. Montavon, R. Rüegg, G. Saucy and P.
Zeller, Helv. Chim. Acta, 1956, 39, 2041–2053.
4 J. J. Becker, U. P. Hochstrasser and W. Skorianetz, US Pat.,
3944620, 1972.
5 W. Zhong, L. Mao, Q. Xu, Z. Fu, G. Zou, Y. Li, D. Yin, H. Luo
and S. R. Kirk, Appl. Catal., A, 2014, 486, 193–200.
6 M. Hennig, K. Püntener and M. Scalone, Tetrahedron:
Asymmetry, 2000, 11, 1849–1858.
7 M. Ishihara, T. Tsuneya, H. Shiota, M. Shiga and K. Nakatsu,
J. Org. Chem., 1986, 51, 491–495.
(
Table 1), as well as with the deracemization results of chiral
HIP (Fig. S6†). On the other hand, although additional PELE
calculations showed similar C4-oxo distance for the
R-enantiomer (data not shown), the slightly worse (5 kcal
4
a
−
1
mol ) binding energy of the different pose adopted (with re-
spect to S-4HIP) is in agreement with the S preference experi-
mentally observed (Fig. S6†).
Finally, the dual hydroxylation at the isophorone C and
4
C7 positions by AaeUPO, compared with the selective oxida-
tion at C by CglUPO and rHinUPO (Table 1) was also ana-
4
8 I. Kiran, O. Ozsen, T. Celik, S. Ilhan, B. Y. Gürsu and F.
Demirci, Nat. Prod. Commun., 2013, 8, 59–61.
9 Y. Mikami, Y. Fukunaga, M. Arita, Y. Obi and T. Kisaki,
Agric. Biol. Chem., 1981, 45, 791–793.
lyzed in the PELE simulations. The isophorone C position is
7
not at a catalytically relevant distance in CglUPO (4.4 Å,
Fig. 4B and S8A†), while the C
AaeUPO (3.3 and 2.9 Å, respectively, Fig. 4C and S8B†) are
within reaction limits (the oxo to hydrogen distances are also
7
-oxo and C
4
-oxo distances for
10 I. Kaluzna, T. Schmitges, H. Straatman, D. van Tegelen, M.
Müller, M. Schürmann and D. Mink, Org. Process Res. Dev.,
2016, 20, 814–819.
This journal is © The Royal Society of Chemistry 2018
Catal. Sci. Technol.

View Article Online
Paper
Catalysis Science & Technology
1
1
1
1
1 M. Tavanti, F. Parmeggiani, J. R. G. Castellanos, A. Mattevi
and N. J. Turner, ChemCatChem, 2017, 9, 3338–3348.
2 Y. Wang, D. Lan, R. Durrani and F. Hollmann, Curr. Opin.
Chem. Biol., 2017, 37, 1–9.
3 R. Ullrich, J. Nuske, K. Scheibner, J. Spantzel and M.
Hofrichter, Appl. Environ. Microbiol., 2004, 70, 4575–4581.
4 D. H. Anh, R. Ullrich, D. Benndorf, A. Svatos, A. Muck
and M. Hofrichter, Appl. Environ. Microbiol., 2007, 73,
25 C. Aranda, A. Olmedo, J. Kiebist, K. Scheibner, J. C. del Río,
A. T. Martínez and A. Gutiérrez, ChemCatChem, 2018, 10,
3964–3968.
26 A. Olmedo, J. C. del Río, J. Kiebist, K. Scheibner, A. T.
Martínez and A. Gutiérrez, Chem. – Eur. J., 2017, 23,
16985–16989.
27 E. D. Babot, J. C. del Río, M. Cañellas, F. Sancho, F. Lucas,
V. Guallar, L. Kalum, H. Lund, G. Gröbe, K. Scheibner, R.
Ullrich, M. Hofrichter, A. T. Martínez and A. Gutiérrez, Appl.
Environ. Microbiol., 2015, 81, 4130–4142.
28 R. Ullrich, M. Poraj-Kobielska, S. Scholze, C. Halbout, M.
Sandvoss, M. J. Pecyna, K. Scheibner and M. Hofrichter,
J. Inorg. Biochem., 2018, 183, 84–93.
29 F. Lucas, E. D. Babot, J. C. del Río, L. Kalum, R. Ullrich, M.
Hofrichter, V. Guallar, A. T. Martínez and A. Gutiérrez,
Catal. Sci. Technol., 2016, 6, 288–295.
30 E. D. Babot, J. C. del Río, L. Kalum, A. T. Martínez and A.
Gutiérrez, ChemCatChem, 2015, 7, 283–290.
31 R. Ullrich and M. Hofrichter, FEBS Lett., 2005, 579, 6247–6250.
32 H. Lund, L. Kalum, M. Hofrichter and S. Peter, US Pat.,
9908860B2, 2018.
33 C. R. Otey, Methods Mol. Biol., 2003, 230, 137–139.
34 S. Dezvarei, J. H. Z. Lee and S. G. Bell, Enzyme Microb.
Technol., 2018, 111, 29–37.
5
477–5485.
1
1
5 G. Gröbe, M. Ullrich, M. Pecyna, D. Kapturska, S. Friedrich,
M. Hofrichter and K. Scheibner, AMB Express, 2011, 1,
3
1–42.
6 J. Kiebist, K. U. Schmidtke, J. Zimmermann, H. Kellner, N.
Jehmlich, R. Ullrich, D. Zänder, M. Hofrichter and K.
Scheibner, ChemBioChem, 2017, 18, 563–569.
1
1
7 M. Hofrichter, H. Kellner, M. J. Pecyna and R. Ullrich, Adv.
Exp. Med. Biol., 2015, 851, 341–368.
8 J. E. Stajich, S. K. Wilke, D. Ahren, C. H. Au, B. W. Birren, M.
Borodovsky, C. Burns, B. Canbäck, L. A. Casselton, C. K.
Cheng, J. X. Deng, F. S. Dietrich, D. C. Fargo, M. L. Farman,
A. C. Gathman, J. Goldberg, R. Guigo, P. J. Hoegger, J. B.
Hooker, A. Huggins, T. Y. James, T. Kamada, S. Kilaru, C.
Kodira, U. Kües, D. Kupfert, H. S. Kwan, A. Lomsadze, W. X.
Li, W. W. Lilly, L. J. Ma, A. J. Mackey, G. Manning, F.
Martin, H. Muraguchi, D. O. Natvig, H. Palmerini, M. A.
Ramesh, C. J. Rehmeyer, B. A. Roe, N. Shenoy, M. Stanke, V.
Ter Hovhannisyan, A. Tunlid, R. Velagapudi, T. J. Vision,
Q. D. Zeng, M. E. Zolan and P. J. Pukkila, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 11889–11894.
35 M. Biasini, S. Bienert, A. Waterhouse, K. Arnold, G. Studer,
T. Schmidt, F. Kiefer, T. G. Cassarino, M. Bertoni, L. Bordoli
and T. Schwede, Nucleic Acids Res., 2014, 42, W252–W258.
36 N. Guex, M. C. Peitsch and T. Schwede, Electrophoresis,
2009, 30, S162–S173.
1
9 C. Aranda, R. Ullrich, J. Kiebist, K. Scheibner, J. C. del Río,
M. Hofrichter, A. T. Martínez and A. Gutiérrez, Catal. Sci.
Technol., 2018, 8, 2394–2401.
37 G. M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju and W.
Sherman, J. Comput.-Aided Mol. Des., 2013, 27, 221–234.
38 V. Guallar and F. H. Wallrapp, Biophys. Chem., 2010, 149,
1–11.
39 A. Altun, V. Guallar, R. A. Friesner, S. Shaik and W. Thiel,
J. Am. Chem. Soc., 2006, 128, 3924–3925.
2
2
0 M. Hofrichter and R. Ullrich, Curr. Opin. Chem. Biol.,
2
014, 19, 116–125.
1 A. Olmedo, C. Aranda, J. C. del Río, J. Kiebist, K. Scheibner,
A. T. Martínez and A. Gutiérrez, Angew. Chem., Int. Ed.,
40 D. Lecina, J. F. Gilabert and V. Guallar, Sci. Rep., 2017, 7,
8466.
2
016, 55, 12248–12251.
2
2 E. D. Babot, J. C. del Río, L. Kalum, A. T. Martínez and A.
Gutiérrez, Biotechnol. Bioeng., 2013, 110, 2332.
41 S. Peter, M. Kinne, R. Ullrich, G. Kayser and M. Hofrichter,
Enzyme Microb. Technol., 2013, 52, 370–376.
2
3 A. Gutiérrez, E. D. Babot, R. Ullrich, M. Hofrichter, A. T.
Martínez and J. C. del Río, Arch. Biochem. Biophys.,
42 I. Kaluzna, T. Schmitges, H. Straatman, D. van Tegelen, M.
Müller, M. Schürmann and D. Mink, Org. Process Res. Dev.,
2016, 20, 814–819.
2
011, 514, 33–43.
2
4 S. Peter, M. Kinne, X. Wang, R. Ulrich, G. Kayser, J. T.
43 I. Kaluzna, T. Schmitges, H. Straatman, D. van Tegelen, M.
Müller, M. Schürmann and D. Mink, Org. Process Res. Dev.,
2016, 20, 814–819.
Groves and M. Hofrichter, Rev. Geophys., 2011, 278,
3
667–3675.
Catal. Sci. Technol.
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