Enantiocomplementary Epoxidation Reactions Catalyzed by an Engineered Cofactor-Independent Non-natural Peroxygenase

Article abstract of DOI:10.1002/anie.202001373

Peroxygenases are heme-dependent enzymes that use peroxide-borne oxygen to catalyze a wide range of oxyfunctionalization reactions. Herein, we report the engineering of an unusual cofactor-independent peroxygenase based on a promiscuous tautomerase that accepts different hydroperoxides (t-BuOOH and H₂O₂) to accomplish enantiocomplementary epoxidations of various α,β-unsaturated aldehydes (citral and substituted cinnamaldehydes), providing access to both enantiomers of the corresponding α,β-epoxy-aldehydes. High conversions (up to 98 %), high enantioselectivity (up to 98 % ee), and good product yields (50–80 %) were achieved. The reactions likely proceed via a reactive enzyme-bound iminium ion intermediate, allowing tweaking of the enzyme's activity and selectivity by protein engineering. Our results underscore the potential of catalytic promiscuity for the engineering of new cofactor-independent oxidative enzymes.

Full text of DOI:10.1002/anie.202001373

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Accepted Article
Title: Enantiocomplementary Epoxidation Reactions Catalyzed by an
Engineered Cofactor-Independent Non-Natural Peroxygenase
Authors: Guangcai Xu, Michele Crotti, Thangavelu Saravanan, Kim
Kataja, and Gerrit J. Poelarends
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202001373
Angew. Chem. 10.1002/ange.202001373
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http://dx.doi.org/10.1002/ange.202001373

10.1002/anie.202001373
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Enantiocomplementary Epoxidation Reactions Catalyzed by an
Engineered Cofactor-Independent Non-Natural Peroxygenase
Guangcai Xu,[a] Michele Crotti,[a] Thangavelu Saravanan,[a,b] Kim M. Kataja[a] and Gerrit J.
Poelarends*[a]
[a]
G. Xu, Dr. M. Crotti, Dr. T. Saravanan, K. M. Kataja, Prof. Dr. G. J. Poelarends
Department of Chemical and Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[b]
Present address: School of Chemistry, University of Hyderabad
P.O. Central University, Gachibowli, Hyderabad 500046 (India)
Supporting information for this article is given via a link at the end of the document.
Abstract: Peroxygenases are heme-dependent enzymes that use
peroxide-borne oxygen to catalyze wide range of
oxyfunctionalization reactions. Herein, we report the engineering of an
unusual cofactor-independent peroxygenase based on
Our results showcase the potential of catalytic promiscuity for the
creation of new cofactor-independent oxidative enzymes.
a
We have recently demonstrated that 4-OT can catalyze the
Michael addition of nitromethane to cinnamaldehyde via an
enzyme-bound iminium ion intermediate, which is formed
between cinnamaldehyde and the catalytic active site Pro-1
residue.[7] In this study, we examined if this catalytic mechanism
of 4-OT could be exploited for other useful synthetic applications,
and envisioned that substituting nitromethane with t-BuOOH
could trigger a C-O bond formation between the reactive enzyme-
bound iminium ion species and t-BuOOH (Scheme S1). The
resulting enamine intermediate may undergo ring closure to
construct the final epoxide moiety. To our delight, when wild-type
4-OT (0.33 mg/mL) was incubated at room temperature with 200
mM t-BuOOH (1, Figure 1) and 1 mM cinnamaldehyde (2a) in 20
mM sodium phosphate buffer (pH 7.3) containing 5% (v/v) EtOH,
38% of substrate 2a was consumed within 48 h and the
corresponding α,β-epoxy-aldehyde 3a was identified by GC-MS
analysis. Chiral HPLC analysis of the alcohol derivative of 3a
revealed the product to be (2R,3S)-3a with an e.r. of 62:38 and a
d.r. of 92:8 (syn/anti). The S configuration of the C3 carbon of 3a
indicates that t-BuOOH selectively attacks cinnamaldehyde from
the re-face, similar to what was previously observed for the
nitromethane addition reaction.[7] These results demonstrated that
4-OT has low-level promiscuous peroxygenase activity and may
possibly serve as a template to construct a cofactor-independent
peroxygenase.
Motivated by these initial findings, a mutability-landscape-
guided protein engineering approach[8] was applied to enhance
the promiscuous peroxygenase activity of wild-type 4-OT. For this,
a previously constructed collection of 4-OT genes encoding
nearly all possible single-mutant variants of 4-OT[9] was screened
to identify hotspot positions in the protein where mutations give
improved peroxygenase activity. Improved variants were
identified by monitoring the depletion of substrate 2a in a
spectrophotometric kinetic assay in multiwell plates (see
Supporting Information). Interestingly, six mutations at position
F50 significantly improved (>5-fold) the peroxygenase activity,
with F50A (10-fold enhanced activity) and F50V (25-fold
enhanced activity) being the best mutant enzymes (Figure 1).
Several mutations at positions Q4, I41, M45 and H49 also
resulted in enzyme variants with 2- to 5-fold enhanced activity
compared to wild-type 4-OT, including mutants Q4Y, I41Y, M45I
a
promiscuous tautomerase that accepts different hydroperoxides (t-
BuOOH and H2O2) to accomplish enantiocomplementary
epoxidations of various α,β-unsaturated aldehydes (citral and
substituted cinnamaldehydes), providing access to both enantiomers
of the corresponding α,β-epoxy-aldehydes. High conversions (up to
98%), high enantioselectivity (up to 98% ee), and good product yields
(50-80%) were achieved. The reactions likely proceed via a reactive
enzyme-bound iminium ion intermediate, allowing tweaking of the
enzyme’s activity and selectivity by protein engineering. Our results
underscore the potential of catalytic promiscuity for the engineering of
new cofactor-independent oxidative enzymes.
Peroxygenases are oxidative enzymes catalyzing the
insertion of an oxygen atom into a variety of substrates using
peroxide-borne oxygen instead of dioxygen.[1] Earlier studies
have mainly focused on the peroxygenase activity of
chloroperoxidases that normally catalyze peroxide-driven
halogenation of organic compounds[2] and engineered
cytochrome P450 monooxygenases that are able to promote
peroxide-mediated hydroxylation reactions.[3] However, since the
discovery of the unspecific peroxygenase AaeUPO4 in 2004,[4]
which uses hydrogen peroxide to catalyze diverse
oxyfunctionalization reactions including oxidations, sulfoxidations,
epoxidations and hydroxylations,[5] there has been an increasing
interest to discover and develop novel peroxygenases. Note that
peroxygenases are cofactor-dependent enzymes, typically relying
on heme to react with hydrogen peroxide, forming an oxoferryl-
heme as the actual oxygenating species to promote
oxyfunctionalization reactions.[1,5a,6]
In this study, we report the engineering of an unusual
cofactor-independent peroxygenase based on the promiscuous
peroxygenase activity of the enzyme 4-oxalocrotonate
tautomerase (4-OT). This engineered non-natural peroxygenase
accepts different hydroperoxides (t-BuOOH and H2O2) to achieve
enantiocomplementary epoxidations of various α,β-unsaturated
aldehydes, providing access to both enantiomers of the resultant
α,β-epoxy-aldehydes with high enantiomeric excess (up to 98%).
1
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and H49T. The most beneficial mutations, F50V and F50A, were
subsequently combined with mutations at other hotspot positions
by using site-saturation mutagenesis (Table S2). Activity
screening of the seven mutant libraries resulted in the
identification of three double mutants (Q4Y/F50V, Q4F/F50A and
M45I/F50A) with around 1.6- to 3.5-fold enhanced activity
compared to the parental enzymes. Based on these results, the
unsaturated aldehydes using t-BuOOH as oxidant. The results
showed that 4-OT(YIA) has a broad substrate scope, accepting
ortho-, meta- and para-substituted cinnamaldehydes (2a-i, Figure
2), and catalyzes their epoxidation with good conversions (80-
97%) to yield the corresponding products (3a-i) with excellent
enantiopurity (e.r. up to 99:1). The d.r. values (up to 92:8)
obtained indicate that the enzyme has good stereocontrol over
both carbons of the double bond of 2. The enzyme also accepts
the aliphatic α,β-unsaturated aldehyde citral (2j) with good
enantioselectivity, moderate diastereoselectivity, and excellent
regioselectivity (Figure 2). All epoxide products have the syn
configuration with 2R,3S being the major enantiomer, indicating
that t-BuOOH exclusively attacked from the re-face of the
enzyme-bound iminium ion intermediate (Figure 3). Note that
while the enzymatic α,β-epoxy-aldehyde products 3 have a 2R,3S
configuration, the corresponding α,β-epoxy-alcohols have a
2S,3S configuration. This deviant configuration of the α,β-epoxy-
alcohols is due to different prioritization of the substituents at the
C2 chiral center relative to their α,β-epoxy-aldehyde precursors.
Given that t-BuOOH is a relatively large hydroperoxide, we
investigated if the use of a small hydroperoxide such as H2O2
would change the preference of attack to the si-face (Figure 3).
Again, compound 2a was used as a substrate for initial testing
with H2O2 as oxidant and mutant 4-OT(YIA) as catalyst. Notably,
the epoxidations with H2O2 showed relatively high non-enzymatic
(background) reaction rates when applying the same conditions
as used with t-BuOOH. To suppress the background reaction,
without diminishing the enzymatic activity, the pH of the reaction
was lowered from 7.3 to 6.3 (Figure S1). Gratifyingly, the results
showed that the use of H2O2 not only inverted the stereochemistry
of the reaction, but product 3a was also obtained with good e.r.
(92:8) and d.r. (84:16) values (Figures 2 and 3). Thus, an
enantiocomplementary epoxidation of 2a was achieved by
replacing t-BuOOH by H2O2 as the oxidant. Next, we evaluated
the same set of α,β-unsaturated aldehydes 2b-2j for 4-OT(YIA)-
catalyzed epoxidation reactions using H2O2 (Figure 2).
Importantly, the stereo inversions occurred with all aromatic
substrates (2a-2i), and high conversions and good enantio- and
diastereoselectivity were observed. Notably, the epoxidation
reaction with the aliphatic substrate citral (2j) also showed an
inversion of diastereoselectivity when using H2O2 instead of t-
BuOOH, resulting in the anti-product as the major isomer; the syn-
product also showed an inverted enantiomer ratio (Figure S30).
Having achieved enantiocomplementary epoxidation
reactions, we next investigated semi-preparative scale
chemoenzymatic synthesis of α,β-epoxy-alcohols 4a-j using 4-
OT(YIA) as catalyst. With t-BuOOH as oxidant, the reactions were
performed with higher substrate concentrations (50 mM 2a, 20
mM 2b-g & 2j, 5 mM 2i), maintaining high conversions and
stereoselectivity with good isolated product yields (Table 1). Only
a minor decrease in enantio- and diastereoselectivity was
observed, most likely due to the non-enzymatic background
reaction, since somewhat longer reaction times were needed. The
semi-preparative scale synthesis using H2O2 as oxidant was also
attempted using 20 mM 2a and 22 mM H2O2. However, it was
observed that the e.r. of the product (isolated as 4a) substantially
decreased to 73:27 (92:8 when using 1 mM 2a, Figure 2). We
further explored this unexpected result by testing different
concentrations of 2a and H2O2 in analytical-scale reactions (Table
S3). The results revealed that an increasing concentration of 2a
has a negative effect on the e.r. of the final product 4a, while the
mutant
libraries
Q4X/M45I/F50A,
Q4Y/M45X/F50A,
Q4F/M45X/F50A, and Q4Y/M45I/F50X were constructed (Table
S2). Screening of these four libraries led to the discovery of the
triple mutant Q4Y/M45I/F50A (YIA) that showed a remarkable 60-
fold enhancement in activity for the epoxidation of 2a compared
to the wild-type enzyme (Figure 1). Notably, this mutant enzyme
also has enhanced enantioselectivity, allowing the production of
(2R,3S)-3a with high enantiopurity (e.r. = 98:2). Replacement of
Pro-1 with an alanine in 4-OT(YIA) led to a 210-fold decrease in
t-BuOOH-dependent epoxidation of 2a (Figure S31), providing
support for a reaction mechanism that proceeds via iminium ion
formation between the active site Pro-1 residue and the α,β-
unsaturated aldehyde substrate (Scheme S1).
Figure 1. Peroxygenase activity of 4-OT variants. (A) Reaction scheme of 4-OT
catalyzed epoxidation of 2a using t-BuOOH as oxidant. (B) Comparison of the
peroxygenase activity of wild-type 4-OT (WT) and engineerd 4-OT variants. (C)
Reaction progress curves using different 4-OT variants. The initial rate of the 4-
OT(YIA)-catalyzed reaction corresponds to 16 µM/min; specific activity is 48
mU/mg of protein.
With the improved 4-OT mutant YIA in hand, the substrate
scope of this enzyme was explored by testing a set of α,β-
2
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10.1002/anie.202001373
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concentration of H2O2 has no effect (Table S3). This effect may
be caused by the higher concentration of peroxyhydrate (S4)
formed between the enzymatic aldehyde product (3a) and H2O2
when higher substrate (2a) concentrations were used (Scheme
In reactions with t-BuOOH, the peroxyhydrate (S5) of the
enzymatic aldehyde product (3a) is not a hydroperoxide, and
therefore can not be accepted by the enzyme as an oxidant.
Consequently, the negative effect of high substrate
concentrations on the enantiopurity of the final product was not
observed in 4-OT(YIA)-catalyzed epoxidation reactions with t-
BuOOH (Scheme S2).
S2). This peroxyhydrate can be considered as
a large
hydroperoxide that, like t-BuOOH, attacks the iminium ion on the
re-face during the reaction, thus lowering the e.r. of the product.
Figure 2. Substrate scope of the epoxidation reactions catalyzed by 4-OT(YIA) (Q4Y/M45I/F50A) using t-BuOOH or H2O2 as the oxidant. Reaction conditions: 2 (1
mM), t-BuOOH (100 mM) or H2O2 (25 mM), 4-OT(YIA) (0.1 mg/mL), NaPi buffer (20 mM), 5% (v/v) EtOH (MeCN for 2f, 2g and 2h). Conversions were calculated
based on the depletion of the absorbance corresponding to substrate 2. Product identifications were performed by GC-MS analysis. The enzymatic α,β-epoxy-
aldehyde products 3 were reduced to the corresponding α,β-epoxy-alcohols with NaBH4 for chiral HPLC analysis. The e.r., d.r. and absolute stereochemistry of
products were determined by chiral HPLC using authentic standards. The diastereomer ratio (d.r.) is defined as the syn:anti ratio. 2j: E/Z = 3:2; n.d.: not determined.
3
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Figure 3. Enantiocomplementary epoxidation of 2a catalyzed by 4-OT(YIA) using t-BuOOH or H2O2 as oxidant. The predominant enzymatic diastereomer has the
syn configuration; the minor anti diastereomeric product presumably is formed by rotation of the C2-C3 bond after addition of the peroxide, but before ring closure.
In summary, we have exploited the catalytic promiscuity of
4-OT to engineer an effective peroxygenase (4-OT-YIA) that
promotes peroxide-driven asymmetric epoxidations of α,β-
unsaturated aldehydes. Unlike the peroxygenases offered by
nature, the engineered non-natural peroxygenase 4-OT(YIA)
does not require any cofactors, such as heme, for catalysis. While
Candida antarctica lipase B (CALB) reportedly performs cofactor-
independent epoxidation reactions, the described enzymatic
products were either racemic[10] or reported without demonstrating
any enantioselectivity[11]. Notably, several amino acid and peptide
organocatalysts, as well as bovine serum albumin (BSA), have
been shown to catalyze asymmetric peroxide-driven epoxidations
of α,β-unsaturated aldehydes and ketones, but these systems
offer a more general mode of catalysis and, with the exception of
BSA, often require the use of organic solvents.[12] In contrast,
peroxygenase 4-OT(YIA) uses a distinct active-site process to
accomplish highly enantioselective epoxidations of α,β-
unsaturated aldehydes, and by using either t-BuOOH or H2O2 to
drive the reaction, access to both product enantiomers can be
achieved. This may suggest that there are two binding pockets for
hydroperoxides in the active site of 4-OT(YIA); one relatively large
and hydrophobic pocket at the re-face of the enzyme-bound
iminium ion that preferably binds t-BuOOH and one relatively
small and probably more hydrophilic pocket at the si-face of the
iminium ion that selectively recruits H2O2 for asymmetric
epoxidations (Figure 3). We have initiated structural studies of 4-
OT(YIA) aimed at elucidating the mechanistic origin of the
enantiocomplementary epoxidation reactions. Furthermore, given
that 4-OT(YIA) exhibits a moderate specific activity of 48 mUꢀmg−1
for the t-BuOOH-driven epoxidation of 2a, current work in our
group is focused on further improving the peroxygenase activity
of this promising cofactor-independent enzyme, making use of a
recently developed selective colorimetric "turn-on" probe for
efficient engineering of iminium biocatalysis.[13] The non-natural
peroxygenase activity of 4-OT, together with its previously
reported promiscuous C-C bond-forming Michaelase[7,9,14] and
aldolase[15] activities, emphasize the chemical versatility of the 4-
OT protein scaffold.
Table 1. Preparative-scale 4-OT(YIA)-catalyzed epoxidations of α,β-unsaturated aldehydes using t-BuOOH as oxidant.[a]
Conv.
Abs.
Entry
2
R1
R2
t [h]
e.r.[c]
d.r.[d]
(Yield[%])[b]
93 (52)
85 (55)
85 (68)
91 (62)
95 (50)
98 (80)
92 (64)
90 (61)
79 (50)
config.[e]
1[f]
2
a
b
c
d
e
f
H
H
Ph
o-Cl-Ph
24
48
66
48
24
46
48
45
96
98:2
93:7
94:6
97:3
96:4
98:2
97:3
97:3
96:4
92:8
68:32
85:15
89:11
83:17
92:8
2S,3S
2S,3S
2S,3S
2S,3S
2S,3S
2S,3S
2S,3S
2S,3S
2S,3S
3
H
m-Cl-Ph
4
H
p-Cl-Ph
5
H
p-F-Ph
6[f]
7
H
p-Br-Ph
g
i
H
p-NO2-Ph
o-Me-Ph
89:11
80:20
60:40
8
H
9[g]
j
Me
(CH3)2CCHCH2CH2
4
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[a] General reaction conditions: 2 (20 mM), t-BuOOH (100 mM), 4-OT(YIA) (0.93 mg/mL) in 20 mM NaPi (sodium phosphate, pH 7.3) buffer with 10% (v/v) EtOH
(MeCN for 2f & 2g), volume = 10 mL (5 mL for 2a, 40 mL for 2f), enzymatic product 3 reduced with NaBH4 to 4 after completion of the reaction [b] Conversion
determined by GC-MS; yield of the diastereomer mixture. [c] syn diastereomer; determined by chiral HPLC. [d] syn/anti. [e] syn diastereomer; determined by
comparison with authentic standards on chiral HPLC. [f] conc. 2a = 50 mM, conc. 2f = 5 mM, see supporting information for details. [g] 2j: E/Z = 3:2; 1.3 mg/mL 4-
OT(YIA).
2016, 1225–1228; c) M. Rahimi, E. M. Geertsema, Y. Miao, J.-Y. van der
Meer, T. van den Bosch, P. de Haan, E. Zandvoort, G. J. Poelarends,
Org. Biomol. Chem. 2017, 15, 2809–2816.
Acknowledgements
We acknowledge financial support from the Netherlands
Organization of Scientific Research (VICI grant 724.016.002 and
ECHO grant 713.015.003).
Keywords: oxidative enzyme • peroxygenase • epoxidation •
peroxide • enzyme engineering
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5
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Entry for the Table of Contents
No cofactor needed! An uncommon cofactor-independent peroxygenase was engineered based on a promiscuous tautomerase that
uses different hydroperoxides (t-BuOOH and H2O2) to achieve enantiocomplementary epoxidations of various α,β-unsaturated
aldehydes, giving access to both enantiomers of the corresponding α,β-epoxy-aldehydes with high enantiopurity (up to 98% ee).
6
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