Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution

Article abstract of DOI:10.1021/jacs.8b04742

Controlling the regioselectivity of Baeyer-Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer-Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

Full text of DOI:10.1021/jacs.8b04742

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Article
Overriding Traditional Electronic Effects in Biocatalytic
Baeyer-Villiger Reactions by Directed Evolution
Guangyue Li, Marc Garcia-Borràs, Maximilian J. L. J. Fürst, Adriana
Ilie, Marco W. Fraaije, Kendall N. Houk, and Manfred T. Reetz
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 25 Jul 2018
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Journal of the American Chemical Society
Overriding Traditional Electronic Effects in Biocatalytic
Baeyer-Villiger Reactions by Directed Evolution
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Guangyue Li1,2,3, Marc GarciaꢀBorràs4, Maximilian J. L. J. Fürst5, Adriana Ilie2,3, Marco W. Fraaije5, K. N. Houk4,* and
Manfred T. Reetz2,3,
*
1State Key Laboratory for Biology of Plant Diseases and Insect Pests/Key Laboratory of Control of Biological Hazard Factors
(Plant Origin) for Agriꢀproduct Quality and Safety, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of
Agricultural Sciences, Beijing 100081, China.
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2MaxꢀPlanckꢀInstitut für Kohlenforschung, KaiserꢀWilhelmꢀPlatz 1, 45470 Mülheim, Germany.
3Department of Chemistry, PhilippsꢀUniversity, HansꢀMeerweinꢀStrasse 4, 35032 Marburg, Germany.
4Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States.
5Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
ABSTRACT: Controlling the regioselectivity of BaeyerꢀVilliger（BV）reactions remains an ongoing issue in organic chemistry, be
it by synthetic catalysts or enzymes of the type BaeyerꢀVilliger monooxygenases (BVMOs). Herein, we address the challenging
problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates which
are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding
pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4ꢀphenylꢀ2ꢀbutanone as
substrate, the regioselectivity was reversed from 99:1 (wildꢀtype enzyme in favor of the normal product undergoing 2ꢀphenylethyl
migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective
reaction using the synthetic reagent mꢀCPBA which provides solely the normal product. Reversal of regioselectivity was also
achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the
evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity
in reactions of 4ꢀphenylꢀ2ꢀbutanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that
the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and
transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a
reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally
causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein
engineering of BVMOs.
undergoes fragmentation and rearrangement to the product. The
1. INTRODUCTION
control of regioselectivity in the reaction of unsymmetrical
The BaeyerꢀVilliger (BV) reaction, first reported in 1899,1
ketones leading to two different constitutionally isomeric
constitutes a powerful method for converting ketones into the
products continues to be a central synthetic and mechanistic
respective esters or lactones by insertion of an Oꢀatom into a
issue (Scheme 1).2
CꢀC bond adjacent to the carbonyl function.2 Stoichiometric
Stereoelectronic effects play a dominant role in many
amounts of alkylhydroperoxides ROOH or peracids RCO3H
organic transformations,5 BV reactions being a prominent
commonly serve as oxidants, the transformation being
example.2ꢀ4 As shown by a number of computational and
catalyzed by acids, bases, transition metal complexes or
mechanistic studies, these effects play a crucial role in
organocatalysts. When enantioselectivity is involved, as in the
determining the regioselectivity of BV reactions of
desymmetrization of symmetrically substituted cyclic ketones
unsymmetrical ketones.6 The most generally accepted
or oxidative kinetic resolution of racemic ketones, chiral
mechanistic model calls for
a
conformation in the
transition metal catalysts3 or organocatalysts4 have been used
successfully. Mechanistically, oxidants such as ROOH add to
the carbonyl function with formation of a shortꢀlived
tetrahedral species called the Criegeeꢀintermediate, which then
Criegeeꢀintermediate in which the migrating group is
antiperiplanar to the fragmenting peroxy OꢀO bond. This
soꢀcalled primary stereoelectronic requirement enables the
preferred reaction due to an optimal interaction (overlap) of the
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enantiomeric (ꢀ)ꢀtransꢀdihydrocarvone.10 Other examples refer
to reactions of certain βꢀhydroxyꢀ and βꢀaminoketones.11
Abnormal reactivity was also identified in the biosynthesis of
the antibiotic pentalenolactone in which a tricyclic ketone
precursor undergoes a BVMOꢀcatalyzed reaction.12 In all of
these studies, the reasons for the formation of abnormal
products have not been fully elucidated.
Scheme 1. BaeyerꢀVilliger reaction of unsymmetrical acyclic
ketones with potential formation of two different esters.
1
2
3
4
5
6
7
8
R
δ−
site-selective
R1-migration
O
δ+
O
O
OH
R2
R1O
R2
R1
O
Criegee intermediate
R1
R2
9
Protein engineering based on rational design or directed
evolution provides a means to control, inter alia, stereoꢀ and/or
regioselectivity. The first example of BVMO directed evolution
R
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δ−
O
δ+
HO
O
O
R1
OR2
site-selective
R2-migration
involved
the
stereoselective
desymmetrization
of
R1
R2
4ꢀhydroxyꢀcyclohexanone, catalyzed by the cyclohexanone
monooxygenase from Acinetobacter sp. NCIMB 9871
(AcCHMO).13 Later the result was rationalized by QM/MM
CꢀR ơꢀbond with the OꢀO ơ*ꢀorbital. Since the OꢀO is broken
heterolytically as shown in Scheme 1, the best migrating groups
in unsymmetrical ketones are those that stabilize the partial
positive charge best. Therefore, as documented experimentally
many times, the following migratory tendency has been
established: tertꢀBu > Phe ~ IsoꢀPr > Et > Me.2 Thus, in
reactions of unsymmetrical ketones, regioselectivity and
electronic effects are intimately connected. A secondary
stereoelectronic effect has also been postulated in some cases,
which requires the migrating group to be antiperiplanar to one
of the lone electron pairs of the hydroxyl group in the
Criegeeꢀintermediate.6 Exceptions to the generally observed
migratory aptitude with formation of soꢀcalled abnormal
products occur whenever structural peculiarities are involved,
as for example in the case of strained biꢀ or tricylic ketones,2 or
certain cyclic αꢀketoꢀamides.7 Ketones bearing CF3ꢀgroups at
the αꢀposition also react anomalously, which was explained by
DFT computations.8 In the most recent experimental and
computational study, ßꢀketoesters were subjected to BV
reactions using H2O2/BF3, resulting in the formation of stable
Criegee intermediates.6i
14
computations
using the insights gained by a previous
theoretical study concerning the mechanism of the WT as
catalyst in the desymmetrization of 4ꢀmethylcyclohexanone.15
Other protein engineering studies of BVMOs have been
reviewed.16 Whereas the primary focus was on stereoselectivity,
efforts aimed at improving or reversing regioselectivity
(sometimes called siteꢀselectivity) 17 have increased recently. A
seminal example concerns the switch from abnormal to normal
product in the BV reaction of (+)ꢀtransꢀdihydrocarvone
catalyzed by an evolved CHMO mutant from Arthrobacter sp.18
Earlier it had been shown that the reaction of enantiomeric
(ꢀ)ꢀtransꢀdihydrocarvone catalyzed by WT also delivers the
normal product.10 It should be mentioned that in the same
reaction the use of a synthetic Snꢀcontaining zeolite provides
the normal lactone irrespective of the absolute configuration of
transꢀdihydrocarvone,19 which means that in this particular case
the synthetic problem is solved.
Mutations introduced by rational design can also influence
20
regioselectivity in BVMOꢀcatalyzed reactions12,
although
these examples involve strained biꢀ and tricyclic ketones with
special electronic and steric properties. As reported by the
Fraaije group, a unique case of a nonꢀbiased ketone substrate
concerns the reaction of 2ꢀbutanone which was found to result
in a mixture of normal product (ethyl acetate) and abnormal
product (methyl propanoate) in a ratio of ~3:1 at low
conversion using several different CHMOs.21 With the aim of
increasing regioselectivity in favor of the abnormal product, a
semiꢀrational mutagenesis strategy was applied using
AcCHMO.22 A moderate shift to the abnormal product was
induced, from 26% (WT) to 40% (mutant I491A).
Biocatalytic BV oxidations catalyzed by BaeyerꢀVilliger
monooxygenases (BVMOs) constitute an alternative to
synthetic catalysts.9 Nature likewise adheres to the
stereoelectronic effects which govern the migratory aptitude in
synthetic BV reactions, but the protein environment in the
binding pockets of these enzymes may lead to the formation of
abnormal products or product mixtures, depending upon which
BVMO is used. Examples of abnormal product formation
involve BVMOꢀcatalyzed reactions of terpenones such as
cisꢀdihydrocarvone,
carvomenthone
and
menthone.9
Interestingly, one study found that (+)ꢀtransꢀdihydrocarvone
also leads to the abnormal product, in contrast to the
Scheme 2. BaeyerꢀVilliger oxidation of model ketones 1, 4 and 7
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unconventional variation of our previously developed
catalyzed by TmCHMO used in this study.
1
semiꢀrational directed evolution technique based on
Combinatorial ActiveꢀSite Saturation Test (CAST) and
Iterative Saturation Mutagenesis (ISM) at sites lining the
binding pocket,23 flanked by protein QM/MM computations.24
O2
H2O
TmCHMO
2
3
4
5
6
7
8
9
O
O
O
O
O
O
NADP+
NADPH
1
2
3
O2
H2O
TmCHMO
2. RESULTS AND DISCUSSION
O
O
O
NADP+
O
NADPH
2.1
Directed
Evolution
Experiments
and
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Characterization of Mutants. As the model BVMO in this
study, we chose the recently discovered thermostable
cyclohexanone monooxygenase from Thermocrispum
municipal DSM 44069 (TmCHMO), because 1) a crystal
structure was available, and 2) it had been shown that it
displays similar kinetic properties when compared with
AcCHMO, i.e., it has sufficient activity at room temperature.25
In an initial directed evolution study of this enzyme, we had
already applied CAST/ISM, in that case with the aim of
inverting enantioselectivity in the desymmetrization of
4ꢀmethylcyclohexanone.26 By first performing NNKꢀbased
saturation mutagenesis individually at 11 selected residues
lining the binding pocket (L145, L146, F248, F279, R329,
F434, T435, N436, L437, W492 and F507) as already reported
in our previous study 26, 5 hotspots were identified on the basis
of the stereoselectivity criterion. Mutant L437A proved to be
the best one. This mutant was then employed as the template for
NDTꢀbased saturation mutagenesis at the remaining four
hotspots in the form of two randomization sites, A (F434/T435)
and B (L146/F507) and an ISM scheme leading to reversal of
enantioselectivity.26 Anticipating that in the present case
reversal of regioselectivity would be more difficult, we
developed a different strategy. We first docked substrate 1 into
the crystal structure of TmCHMO (Fig. 1) and found that all 11
previously selected amino acid positions remain aligned to the
binding pocket, i.e, the binding process does not cause major
conformational changes of these residues. Therefore, we
screened by automated GC the already available original 11
miniꢀNNK libraries26 in the BV transformations of all three
ketones 1, 4 and 7 (Scheme 2), but subsequently tested a
technique not reported previously. The chemical reactions
using mꢀCPBA as oxidant led to exclusive or nearly exclusive
formation of the normal products 2 (> 99:1), 5 (> 99:1), and 8
(95:5) (Fig. S4、S10 and S16).
O
O2
H2O
TmCHMO
O
O
O
O
NADP+
NADPH
9
7
In the present study we addressed the problem of
switching normal to abnormal BVMO regioselectivity using
three linear ketones, 1, 4 and 7 (Scheme 2). This is particularly
challenging because, unlike the above mentioned ketones, 9ꢀ11,18
these linear substrates are not structurally biased. The plan was
to introduce effective point mutations by directed evolution,
flanked by a theoretical study in the quest to unveil the
structural and mechanistic reasons for such a switch. Upon
testing several WT BVMOs or synthetic reagents in the BV
reaction of these ketones, complete or extremely high
preference for the normal products 2, 5 and 8, respectively, was
observed. We anticipated that laboratory evolution of BVMO
mutants was necessary that override the traditional
stereoelectronic effect. Experimentally, we applied an
Figure 1. TmCHMO structure model showing docked
4ꢀphenylbutanꢀ2ꢀone (1) (in cyan) based on the crystal structure of
wildꢀtype TmCHMO (pdb code 5M10),25 and the 11 CAST amino
acids lining 1 displayed in purple. Green: NADP+; Yellow: FAD.
In the case of ketone 1, several variants were identified by
this initial exploration that showed moderately decreased
selectivity for the normal product 2, namely L145A, L145G,
L145V, L437T and L437A, thus pointing the way towards
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possible reversal of regioselectivity. The best hit proved to be 2ꢀresidue sites (A, B, C and D). This requires in each case the
1
2
3
4
5
6
7
8
mutant L145G, which was chosen as a template to visit position
L437, leading to two variants with reversed regioselectivity,
LGY2ꢀB12 (L145G/L437T, 2:3 = 17:83) and LGY2ꢀD8
screening of 4 microtiter plates for 95% coverage assuming the
absence of bias, and NNK for the single amino acid library E
one plate only. Several mutants were discovered showing
significantly enhanced regioselectivity favoring the abnormal
(L145G/L437V, 2:3
=
23:77). Thereafter, LGY2ꢀB12
(L145G/L437T) was used as a template to test ISM by
considering the remaining 9 residues (L146, F248, F279, R329,
F434, T435, N436, W492 and F507), which were grouped into
five randomization sites: A (F279, F507), B (L146, N436), C
(F248, W492), D (F434, T435) and E (R329). In contrast to our
earlier stereoselectivity study,26 this meant that residues were
considered that were not hotspots in the initial 11 exploratory
NNKꢀbased mutagenesis experiments. In order to minimize the
screening effort, NDT codon degeneracy encoding only 12
amino acids (Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, Arg,
Ser and Gly) was chosen for constructing libraries at the
product
3
in
library
D,
namely
LGY3ꢀDꢀA9
(L145G/F434I/T435I/L437T, 2:3
(L145G/F434G/T435F/L437T, 2:3
=
3:97), LGY3ꢀDꢀE1
2:98), LGY3ꢀDꢀB7
9
=
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(L145G/F434N/T435G/L437T, 2:3 = 8:92) and LGY3ꢀDꢀE9
(L145G/F434G/T435Y/L437T, 2:3 = 7:93). The libraries
created at the other four sites did not harbor any positive
variants (Table S1). The best results are summarized in Figure
2a.
An analogous procedure was applied to ketone 4, the best
results being summarized in Figure 2b (see also Table S2 for
Figure 2. Iterative saturation mutagenesis (ISM) exploration of TmCHMO as catalyst in the reactions with focus on reversal of
regioselectivity in favor of the abnormal product using substrate 1 (a), 4 (b), or 7 (c).
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Table 1. Kinetic parameters of WT TmCHMO and best mutants evolved for the abnormal BV product of three substrates.
1
2
Enzyme
Substrate
Mutations
kunc
(
sꢀ1
)
Km (mM)
kcat ( )
sꢀ1
Tm (°C)
3
4
5
6
7
8
9
WT
1
1
1
4
4
7
7
7
7
ꢀ
0.016 ± 0.0030
0.20 ± 0.019
12 ± 7.0
n.d.
0.024 ± 0.0041 0.16 ± 0.0028
0.86 ± 0.060
1.5 ± 0.46
4.4 ± 1.0
5.5 ± 1.7
0.020 ± 0.016
2.8 ± 0.36
0.31 ± 0.11
n.d.
52.67 ± 0.76
50.83 ± 0.76
51.33 ± 0.76
LGY3ꢀDꢀA9
LGY3ꢀDꢀE1
WT
LGY2ꢀB6
WT
LGY1ꢀ492ꢀA7
LGY1ꢀ248ꢀD3
LGY1ꢀ437ꢀE12
L145G/F434I/T435I/L437T
L145G/F434G/T435F/L437T
ꢀ
L437A/W492Y
ꢀ
W492Y
F248D
0.10 ± 0.0096
0.067 ± 0.034
0.067 ± 0.0053
0.015 ± 0.00036
0.029 ± 0.015
0.026 ± 0.0034
0.014 ± 0.0020
0.73 ± 0.014
0.16 ± 0.014
0.37 ± 0.033
0.18 ± 0.026
0.065 ± 0.0048
0.36 ± 0.018
55.33 ± 0.29
53.5 ± 0.5
47.83 ± 2.02
55.33 ± 0.29
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L437A
full details). As can be seen, regioselectivity favoring the
abnormal product 6 was achieved starting from an initial
complete preference by wildꢀtype TmCHMO for the normal
product 5 formed by benzyl migration, but the degree of
reversal remained at the incomplete stage of 5:6 = 30:70.
Nevertheless, in view of the BV reaction using mꢀCPBA which
occurs with exclusive benzyl migration, this is a synthetically
wildꢀtype enzyme and the most promising mutants with the
corresponding substrates as shown in Table 1 and Figure S20.
Using purified enzymes, we spectrophotometrically measured
the rate of oxidation of the NADPH cofactor at varying
substrate concentrations. The uncoupling rate, namely the
unproductive decay of the peroxyflavin without substrate
oxidation, was measured in control reactions without substrate.
As revealed in Table 1, the uncoupling rate (kunc) was found to
be very low for the WT and most mutant enzymes (0.02ꢀ0.10 sꢀ1)
except for mutant LGY3ꢀDꢀA9 (0.20 sꢀ1), prohibiting accurate
determination of its kcat and Km values for 1. For the WT
enzyme, 4 was found to be the best of the three substrates, with
a kcat of 0.73 sꢀ1 and a Km of 0.86 mM. The evolved mutant
LGY2ꢀB6 showed a reduced kcat of 0.16 sꢀ1, and with a Km of 1.5
mM also a somewhat lower affinity for the substrate than WT
TmCHMO. On the other hand, mutant LGY3ꢀDꢀE1 illustrated a
drastically increased affinity to substrate 1 as compared to WT
(kcat = 0.31 sꢀ1, Km = 12 mM versus kcat = 0.16 sꢀ1/Km = 0.02 mM).
For substrate 7, the mutant LGY1ꢀ492ꢀA7 displayed similar
kinetic parameters relative to WT. LGY1ꢀ248ꢀD3 had a very
low Km (0.02 mM), but also obviously decreased kcat and
probably suffers from substrate inhibition. Clearly the best
mutant is LGY1ꢀ437ꢀE12, showing a slightly lower Km than
WT but the same kcat.
remarkable result
.
In the case of ketone 7, WT TmCHMO proved to be
selective for the normal product 8 produced by preferential
2ꢀphenylethyl migration, but a small amount of the abnormal
product 9 was also formed (8:9 = 85:15). Upon screening the
original 11 miniꢀNNK libraries (see Fig. 2c and Table S3 for
details), it was possible to improve the initial outcome to nearly
exclusive preference for the normal ester (8:9 = 98:2), and also
to completely reversed regioselectivity in favor of the abnormal
product (8:9 = 3:97). Surprisingly, it required less steps to
evolve mutants that allow either benzyl migration or ethyl
migration on an optional basis, in contrast to the outcome of the
reaction of ketone 4 in which benzyl competes with the smaller
methyl group.
Due to the possibility of ester hydrolysis catalyzed by
endogenous lipases or esterases in E. coli cells, the BV
oxidations of the three substrates 1, 4 and 7 were repeated using
purified enzyme of the best TmCHMO mutants. The results
turned out to be the same (Table S4 and Fig. S1ꢀS19), or even
slightly better, e.g., LGY3ꢀDꢀE1 (2:3 = 2:98), LGY2ꢀB6 (5:6 =
26:74), and LGY1ꢀ248ꢀD3 (8:9 = 99:1). A previous report also
noticed a possible effect of substrate concentration on the
regioselectiviy,27 thus we also tested the WT and the mutant
LGY3ꢀDꢀE1 in the conversion of 1 at 50 mM and 100 mM
concentrations. In our system, all reactions retained a high
regioselectivity as shown Table S6, WT leading to 2:3 > 99:1
and LGY3ꢀDꢀE1 leading to 2:3 < 3:97.
To probe thermostability, we measured the melting
temperatures (Tm) of WT and all the evolved mutants, using a
fluorescenceꢀbased thermal shift assay (ThermoFAD).28 It was
found that the thermostability of most mutants is maintained as
compared to WT TmCHMO, except for LGY1ꢀ248ꢀD3
showing a 5 °C drop in Tm (Table 1).
We also tested the best mutant LGY3ꢀDꢀE1 for
semiꢀpreparative scaled transformation of substrate 1 (50 mg)
in 25 mL of reaction volume. Full conversion was achieved
within 24 h, while maintaining the originally evolved high
regioselectivity as analyzed by GC. After isolation using
column chromatography, 46 mg of pure product 3 (83%
To establish whether the mutants are catalytically
competent, we next determined the kinetic parameters for the
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isolated yield) were obtained (Fig. S21ꢀ22).
TS.
1
In order to check whether the present ISM strategy is
accompanied by nonꢀadditive cooperative effects rather than
traditional additive effects,29 we deconvoluted the quadruple
mutant LGY3ꢀDꢀE1 (L145G/F434G/T435F/L437T) and tested
the respective four single mutants L145G, F434G, T435F, and
L437T separately as catalysts in the BV reaction of ketone 1
(Table S5). This procedure revealed dramatically pronounced
cooperative mutational effects, since all four single mutants
were found to show very high or even complete normal
reactivity with formation of ester 2 (L145G: 2:3=81:19; F434G:
2:3=99:1; T435F: 2:3=99:1; L437T: 2:3= 97:3), yet together
they orchestrate opposite regioselectivity with complete
preference for ester 3.
Previously, Thiel and coꢀworkers studied the BV reaction
2
3
4
5
6
7
8
9
mechanism catalyzed by a flavinꢀdependent CHMO using
QM/MM calculations.15 They showed that the enzymatic
reaction proceeds through the formation of a high energy
flavinꢀCriegee intermediate; the migration step is rate and
regioselectivity determining. They also showed the importance
of an active site arginine in stabilizing the negatively charged
Criegee intermediate, although no proton transfer occurs from
the protonated arginine (R329 in the current TmCHMO enzyme)
to the Criegee intermediate; this is different from the peracid
catalyzed mechanism where the protonation of the Criegee
intermediate occurs. 15 More recently, a similar computational
strategy was applied by Huang，Moody and coꢀworkers to study
the BV reaction mechanism in the WT Thermobifida fusca
phenylacetone monooxygenase (PAMO) towards its natural
substrate, phenylacetone, and 2ꢀoctanone.30 They showed, in
line with Thiel studies, the importance of stabilizing the
enzymatic Criegee intermediate and the migration transition
state for enzyme activity.
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2.2 Unraveling the Origin of Regioselectivity by
Computational Modeling. To gain more insights into the
regioselectivity switch through evolution, we used DFT
computations to model the intrinsic preferences of the reaction
and used QM/MM and MD simulations to model the enzymatic
reactions.
Using DFT calculations (see SI for computational details),
we first studied the BV reaction involving substrates 1, 4, and 7
using mꢀCPBA as oxidant. It has been previously reported that
the rateꢀlimiting and also the regioselectivityꢀdetermining step
corresponds to the migration step after the formation of the
protonated Criegee intermediate,6 thus we focused on analyzing
that particular reaction step. Our optimized transition states
(TSs) demonstrated that the intrinsically preferred normal
migration TS is favored over the abnormal one in substrate 1
(ꢁꢁG‡ = ꢀ3.3 kcal·molꢀ1), substrate 4 (ꢁꢁG‡ = ꢀ5.2 kcal·molꢀ1),
and 7 (ꢁꢁG‡ = ꢀ1.9 kcal·molꢀ1), due to the better stabilization of
the partial positive charge generated on the migrating carbon
atom at the TS. The smallest differences in activation barriers
are found for substrate 7 (predicted 8:9 ratio 96:4), which is the
experimentally less selective case when mꢀCPBA is used as
oxidant (95:5). On the other hand, the larger energy barrier
difference favoring the normal migration is found for ketone 4,
the substrate for which protein directed evolution could not
achieve a complete inversion of regioselectivity towards
abnormal product formation (Figure 2b), presumably because
of this intrinsic strong preference towards normal migration of
the benzylic group. These results are in excellent agreement
with the experimental ratios observed when mꢀCPBA is used as
oxidant. The optimized TS structures also highlight the required
antiperiplanar conformation of the migrating group and the
peroxy OꢀO bond (see Fig. S23), needed to reach the SN2ꢀlike
Based on this, we hypothesized that the role played by the
enzyme in controlling the selectivity of the BV reaction may be
due to the imposition of conformational restrictions to the
Criegee intermediate, favoring one particular bound
conformation that could lead to one migration TS while
preventing the other migration from occurring. In principle, one
could expect two relevant conformations to be possible, where
the corresponding migrating groups should be placed
antiperiplanar to the peroxyl OꢀO bond to reach the
corresponding TSs with little further distortion. This hypothesis
implies that new introduced mutations during evolution may
alter the conformation adopted by the Criegee intermediate.
Thus, we analyzed the conformations adopted by the
critical Criegee intermediate when formed in the enzyme active
site in its deprotonated form by using MD simulations. We ran
500 ns MD simulations on the Criegee intermediate bound into
the different enzyme variants and monitored the dihedral angles
formed
by
the
O(peroxy1)ꢀO(peroxy2)ꢀC(carbonyl)ꢀC(migrating)
atoms
during the trajectories. A dihedral angle value close to ±180º
indicates that the migration is possible because the considered
migrating group is antiperiplanar to the OꢀO peroxy bond in
that bound conformation. We chose the bulkiest ketone 1 for
our computational modeling because experimentally it exhibits
the largest degree of selectivity (Fig. 2a).
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When the Criegee intermediate is formed, a new
quaternary carbon center is generated. We considered the
possibility of forming both R and S stereoisomers when the
peroxyflavin addition to ketone 1 occurs. Based on the docking
results (see Fig. 1) and on MD simulations carried out (see Fig.
S24), we concluded that only the RꢀCriegee stereoisomer may
be formed in order to keep a less strained conformation of the
phenylethyl group and the strong Hꢀbonding interaction with
R329 needed for stabilizing the negative charge generated on
the former carbonyl oxygen atom. MD simulations for the
1ꢀRꢀCriegee intermediate bound into the WT BVMO showed
that it mainly explores one conformation in which the dihedral
angle corresponding to the phenylethyl group migration
(dihed1ꢀnormal) is ca. 150ꢀ180º during the entire simulation
time, as shown in Figure 3a. The equivalent dihedral angle for
the methyl migration (dihed1ꢀabnormal) is ca. ꢀ60 toꢀ100º.
Based on this, only the phenylethyl migration would be
possible since the conformation that the Criegee intermediate
adopts in the enzyme active site only allows the phenylethyl
group to be antiperiplanar to the peroxyl OꢀO bond, which is
required for the migration to occur. This group is also most
effective in stabilizing the positive charge at the peroxy Oꢀatom
in the transition state, in line with the traditional mechanistic
view of normal BV reactions. These results are fully consistent
with the exclusive formation of the normal BV product 2 by the
WT enzyme (Fig. 2a).
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Figure 3. Analysis of 1ꢀRꢀCriegee intermediate conformations
though 500ns MD simulations when bound in the a) WT enzyme;
and b) LGY3ꢀDꢀE1 variant. Black symbols denote selected
snapshots used for further analysis in Figure 4.
because the normal migration is intrinsically preferred
energetically, as early described. Why then, does LGY3ꢀDꢀE1
give the abnormal product?
To analyze this, we carried out QM/MM calculations (see
SI for computational details) to estimate the relative energy
barrier heights for these two possible migrations in the
LGY3ꢀDꢀE1 variant. We selected two representative snapshots
from the MD trajectory where the two different bound
conformations are visited, respectively (see Fig. 3b, and Fig.
4eꢀf). QM/MM calculated energy barriers are 17.5 kcal·molꢀ1
for the methyl migration TS (1ꢀTSꢀabnormal, snapshot at 300ns)
and 20.8 kcal·molꢀ1 for the phenylethyl migration TS
However, when the LGY3ꢀDꢀE1 variant is considered, the
measured dihedral angles for the phenylethyl migration
(dihed1ꢀnormal) and methyl migration (dihed1ꢀabnormal)
indicate that two interconverting conformations of the Criegee
intermediate can be explored (Fig. 3b). From these results, one
may expect to observe the formation of both possible BV
products, although experimentally this variant is highly
selective towards abnormal product formation (Fig. 2a). In
addition, MD simulations on the Criegee intermediate bound
into individual single mutants (L145G, F434G, T435F, and
L437T, see SI Figs. S25 and S26) were carried out. These
simulations showed that some of the mutations introduced,
when considered individually, also allow the intermediate to
explore conformations that may lead to the abnormal migration,
although these single mutants exhibit high selectivities towards
normal product 2 formation. This indicates that the abnormal
product will not be formed just by allowing this conformation
to be explored, presumably
(1ꢀTSꢀnormal, snapshot at 100ns). Thus, although
a
conformation of the Criegee intermediate leading to the normal
BV product 2 (i.e. phenylethyl migration) can be visited, the
higher in energy 1ꢀTSꢀnormal prevents this pathway and favors
instead the formation of the abnormal product 3, through a
much lower energy 1ꢀTSꢀabnormal. The consequence is
preferred methyl migration, although in the traditional view of
BV reactions this group is least effective in stabilizing the
incipient positive charge in the transition state (Scheme 1).
In order to understand the origins of the high energy
barrier for the normal product formation in LGY3ꢀDꢀE1 variant,
we analyzed the equivalent phenylethyl migration TS but in the
WT enzyme using QM/MM (Fig. 4d). In this case, the QM/MM
computed energy barrier is only 14.3 kcal·molꢀ1,
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Figure 4. Active site arrangement in selected snapshots obtained from 500 ns MD trajectories of the 1ꢀRꢀCriegee intermediate bound into the
a) WT enzyme (snapshot at 400 ns in gray); and b) LGY3ꢀDꢀE1 variant (snapshots at 100 ns in purple, and 300 ns in orange). The active sites
are shown from the same perspective. Substrate 1 in the 1ꢀRꢀCriegee intermediate is shown in blue. c) Superimposition of the WT (snapshot
at 400 ns in gray) and LGY3ꢀDꢀE1 (snapshot at 300 ns in orange) 1ꢀRꢀCriegee intermediate bound active sites. QM/MM optimized transition
state geometries (only atoms included in the QMꢀregion are shown) for selected snapshots obtained from d) WT 1ꢀTSꢀnormal phenylethyl
migration (snapshot at 400 ns); e) LGY3ꢀDꢀE1 1ꢀTSꢀnormal phenylethyl migration (snapshot at 100 ns); and f) LGY3ꢀDꢀE1 1ꢀTSꢀabnormal
methyl
migration
(snapshot
at
300
ns).
Energies
are
given
in
kcal·molꢀ1,
distances
in
Å,
and
O(peroxy1)ꢀO(peroxy2)ꢀC(carbonyl)ꢀC(migrating) dihedral angles in degrees. g) Superimposition of the 1ꢀRꢀCriegee intermediate QM/MM
optimized 1ꢀTSꢀnormal structures (only Criegee intermediate atoms are shown) in the WT (snapshot at 400 ns in gray) and LGY3ꢀDꢀE1
(snapshot at 100 ns in purple).
being more than 5 kcal·molꢀ1 lower. When comparing the
optimized TS geometries (Criegee intermediate atoms included
in the QM region, Fig. 4g), it is found that the phenylethyl
migrating group in LGY3ꢀDꢀE1 is more distorted than in the
WT TS due to the new conformation that the Criegee
intermediate is forced to adopt in LGY3ꢀDꢀE1. The
LGY3ꢀDꢀE1 1ꢀTSꢀnormal is a late TS as compared to the WT
1ꢀTSꢀnormal and the LGY3ꢀDꢀE1 1ꢀTSꢀabnormal (Fig. 4dꢀf),
where the main differences arise from the breaking CꢀC bond
distances (1.93Å vs. 1.77Å and 1.76Å, respectively).
with the FAD cofactor, displacing it from its original position.
The methyl group of L437T side chain can also effectively
create new CHꢀπ interactions with the phenyl ring of the
Criegee intermediate further stabilizing this conformation.
Finally, F434G and T435F mutations allow W492 to move
closer to the Criegee intermediate phenyl ring, pushing it down,
and forcing it to keep that particular orientation. All these
effects increase the strain on the phenylethyl group when it is
migrating, resulting in a worse orbital overlap and a
destabilization of this normal migration TS. On the other hand,
the new conformation that the Criegee intermediate explores,
allows the methyl migration to form the abnormal BV product 3.
Consequently, evolutionary pressure enables the LGY3ꢀDꢀE1
enzyme to stabilize a bound conformation and a TS that could
efficiently lead to the abnormal product formation. And more
importantly, the new introduced set of mutations also
A closer look into the enzyme active site revealed the
profound changes cooperatively induced by the set of mutations
introduced by directed evolution. As shown in Fig. 4aꢀc, L145G
and L437T mutations create more space to accommodate the
phenyl ring in this region of the active site pocket. At the same
time, the side chain of the new introduced L437T is Hꢀbonding
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significantly destabilizes the intrinsically preferred migration A model for explaining the molecular phenomenon behind
1
pathway by increasing the barrier of the normal migration TS
due to cooperative effects of the different mutated residues.
These prevent the normal migration TS from occurring, leading
to the regioselectivity observed with LGY3ꢀDꢀE1.
Simulation studies of the reactions of phenylacetone
substrate 4 catalyzed by the WT and mutant LGY2ꢀB6 are
reported in the SI.
the reversal of regioselectivity was constructed by MD
simulations and QM/MM computations. The mutations
introduced by directed evolution result in changes in the
conformation of the respective Criegee intermediates in the
binding pocket of the enzyme, which allow the abnormal TS to
occur at the same time that largely destabilizes the normal
migration TS, thereby setting the stage for selective
stereoelectronically controlled fragmentation in favor of
abnormal product formation. This novel conformational control
leads to the violation of the traditional rule that those migrating
groups are preferred that stabilize the incipient positive charge
at the peroxy Oꢀatom in the Criegee intermediate most
effectively.
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3. CONCLUSION AND PERSPECTIVES
In summary, we have addressed the challenging issue of
reversing the regioselectivity of
a
BaeyerꢀVilliger
monooxygenase as the biocatalyst in reactions of three
structurally unbiased linear ketones. The switch involves the
preferred formation of the abnormal product resulting from
selective group migrations in the respective Criegee
intermediates, which are normally disfavored due to
stereoelectronic effects. This was accomplished by applying an
unconventional strategy characterized by exploratory saturation
mutagenesis at 11 sites lining the binding pocket (CASTꢀtype
residues), identifying 5 hotspots, and performing iterative
saturation mutagenesis (ISM) at the remaining residues which
were not hotspots in the thermally stable TmCHMO.
Deconvolution of one of the best quadruple mutants with
formation of the respective four single mutants uncovered
strong cooperative mutational effects, not simply additivity as
might be expected.
In conclusion, our work has shown how mutations can
cause distortions of a favored transition state such that a
previously disfavored transition state becomes predominant.
The steric effects that we observed overcame otherwise
favorable stereoelectronic effects. Such a novel mechanism of
control adds another way that enzymes can control selectivity,
and complements the electrostatic factors that Major has shown
can control competing pathways in terpene cyclases.32 We hope
that the mechanistic lessons learned and the insights gained
from our theoretical study will provide a basis for making
future protein engineering of BVMOs easier and faster.
ASSOCIATED CONTENT
Supporting Information. This material is available free of charge
via the Internet at http://pubs.acs.org.”
The experimental results are of synthetic significance
because the standard reagent for BaeyerꢀVilliger oxidations,
mꢀCPBA, was shown to provide the normal products in all three
cases. The evolved mutants suffered essentially no tradeoff in
thermostability and activity as shown by Tm measurements and
kinetics, respectively. In terms of mutagenic “hot spots” in
TmCHMO, our study reveals, inter alia, that position L437
plays an especially critical role in controlling the
regioselectivity in reactions of two of the three model ketones,
but other positions are also of distinct importance. Recently a
review listing hotspots observed in protein engineering of other
BVMOs appeared.31 Adding the present data to this list
provides potentially useful information in further directed
evolution of BVMOs. In future protein engineering efforts,
simply combining some of the known mutations may prove to
be a simple and fast way to obtain selective BVMOs with little
or no highꢀthroughput screening, provided the respective
“rational” choices are guided by structural and theoretical
information.
Materials and chemicals, detailed experimental and computational
protocols, supplementary schemes, tables and figures for the
primers design and libraries screening of TmCHMO, GC analytic
conditions, GC profiles, NMR profiles and structure snapshots
obtained from computation are included in the supporting
information.
AUTHOR INFORMATION
Corresponding Author
* Correspondence should be addressed to (M.T.R.)
reetz@mpiꢀmuelheim.mpg.de , (K.N.H.) houk@chem.ucla.edu.
ACKNOWLEDGEMENT
M.T.R.
acknowledges
generous
support
from
the
MaxꢀPlanckꢀSociety and the LOEWE research cluster
SynChemBio. K.N.H. acknowledges funding from NSF
(CHEꢀ1361104). G.L. thanks the Chinese Academy of Agriculture
Science for the fund of Elite Youth Program and the
MaxꢀPlanckꢀSociety for a postdoc stipend. M.G.ꢀB. thanks the
Ramón Areces Foundation for
a postdoctoral fellowship.
Computational resources were provided by the UCLA Institute for
Digital Research and Education (IDRE) and the Extreme Science
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M.; Fraaije, M. W. Curr. Opin. Chem. Biol. 2010, 14, 138ꢀ144. (h)
and Engineering Discovery Environment (XSEDE), which is
supported by the NSF (OCIꢀ1053575). The research for the work
of M.J.L.J.F. received funding from the European Union (EU)
project ROBOX (grant agreement 635734) under EU's Horizon
2020 Programme Research and Innovation actions H2020ꢀLEIT
BIOꢀ2014ꢀ1.
Leisch, H.; Morley, K.; Lau, P. Chem. Rev. 2011, 111, 4165ꢀ4222. (i)
Hollmann, F.; Arends, I. W. C. E.; Buehler, K.; Schallmey, A.; Bühler,
B. Green Chem. 2011, 13, 226ꢀ265.
(10) Černuchová, P.; Mihovilovic, M. D. Org. Biomol. Chem. 2007,
5, 1715ꢀ1719.
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