“Top” or “bottom” switches of a cyclohexanone monooxygenase controlling the enantioselectivity of the sandwiched substrate

Article abstract of DOI:10.1039/c8cc09951k

Single mutation at the switch residues F432 (F432I/L) or L435 (L435A/G) efficiently reversed the inherent enantiopreference of WT CHMO_Acineto in the Baeyer-Villiger oxidation of various 4-phenyl-cyclohexanone derivatives and 4-alkyl-cyclohexanones, producing a series of substituted lactones with inversed configuration (up to 99% ee and 99% conversion).

Full text of DOI:10.1039/c8cc09951k

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DOI: 10.1039/C8CC09951K
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“Top” or “Bottom” Switches of a Cyclohexanone Monooxygenase
Controlling the Enantioselectivity of the Sandwiched Substrate
Yujing Hu,a, b Jie Wang,a Yixin Cen,a He Zheng,a Meilan Huang,* b Xianfu Lin,a Qi Wu*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Single mutation at the switch residues F432 (F432I/L) or L435 on reversing the stereoselectivity of CHMOAcineto. In the first
(L435A/G) efficiently reversed the inherent enantiopreference of successful case pioneered by Reetz,8a a mutant F432S of
WT CHMOAcineto in the Baeyer–Villiger oxidation of various 4- CHMOAcineto was obtained by screening an error-prone
phenyl-cyclohexanone derivatives and 4-alkyl-cyclohexanones, polymerase chain reaction (epPCR) library that contained more
producing
a series of substituted lactones with inversed than 10,000 variants, with reversed stereoselectivity from 9%
configuration (up to 99% ee and 99% conversion).
ee (R) of WT to 79% ee (S) in the BV oxidation of 4-
hydroxycyclohexanone. The F432S mutant showed impressive
stereoselectivity for various ketones,8b however, except for 4-
hydroxycyclohexanone, the variant only exhibited reversed
stereoselectivity for few substrates.8a Studies on reversing the
enantiopreference of other BVMOs, such as cyclopentanone
monooxygenase (CPMO) and CHMO from Thermocrispum
The tailor-made synthesis of optically pure (R) or (S) lactones
are particularly important because many enantiomerically pure
lactones are highly valuable intermediates for the production of
pharmaceutical drugs1 and chiral polyester biomaterials2.
Hence, development of stereoselective Baeyer–Villiger (BV)
reaction catalysts has atrracted great attention.3,4 Apart from
chiral BV reaction catalysts or reagents developed by chemists,3
Baeyer–Villiger monooxygenases (BVMOs) constitute an
attractive alternative for the synthesis of enantiopure lactones.4
By using BVMOs from different sources, numerous structurally
different ketones can be transformed into corresponding
products with high ee values. One of the biggest challenges in
engineering BVMOs for tailored BV reactions is to inverse the
enantiopreference of BVMOs because BVMOs are usually highly
selective towards one specific configuration of the products.
Cyclohexanone monooxygenase (CHMO) is one of the most
studied BVMOs. CHMO from Acinetobacter sp. NCIMB 9871
(CHMOAcineto),5 as the most prominent member in the CHMO
family, displayed broad substrate scope for substituted
cyclohexanones with relatively high selectivity in comparison to
other well-known BVMOs.4 Directed evolution of CHMOAcineto
has been exploited extensively to achieve the desired
municipal (CHMOThermo
)
have also been reported.10 For
example, one promising quardraple mutant (LGY3-4-E5:
F434I/T435L/L437A/F507V) of CHMOThermo showed highly
reversed selectivity for the BV oxidation of 4-
methylcyclohexanone from 99% ee (S) of WT to 91% ee (R).
However, the reversed selectivity of LGY3-4-E5 mutant toward
4-phenylcyclohexanone was only 30% ee (+) with the
conversion less than 50%,10b and the limitation also laid in the
narrow substrate scope in the attempt to reverse the
enantiopreference. To the best of our knowledge, CHMO
mutants with highly reversed stereoselectivity for a broad scope
of substituted cyclohexanone derivatives, especially for those
with large and bulky substituents (Scheme 1), are not yet
available.
Screening of large libraries created by conventional directed
evolution has been the biggest bottleneck in protein
engineering to achieve admired enantioselectivity.11 Here we
identified two enantioselectivity switches of CHMOAcineto at the
bottom or the top of the sandwiched substrates, under the
guidance of computational simulations. Single mutations at
these two switches obtained from a highly focused library
completely reversed the enantiopreference in the BV reactions
of various 4-phenyl-cyclohexanone derivatives and 4-alkyl-
cyclohexanones, yielding substituted lactones with high
enantioselectivity (up to 99% ee).
properties
such
as
thermostability,6
regio-7
and
stereoselectivity8, substrate scope, heteroatom oxidation and
cofactor usage9. So far, only a few studies have been reported
a. Department of Chemistry, Zhejiang University, Hangzhou 310027 (China).
E-mail: llc123@zju.edu.cn, wuqi1000@163.com
b. School of Chemistry and Chemical Engineering, Queen’s University, Belfast, BT9
5AG (U.K.). E-mail: m.huang@qub.ac.uk
† Electronic Supplementary Information (ESI) available. See
DOI: 10.1039/x0xx00000x
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Journal Name
O
O
View Article Online
DOI: 10.1039/C8CC09951K
g
h
i
j
k
R = 4-ClC6H4
R = n-Pentyl
R = n-Pr
R = Et
R = Me
a
b
c
d
e
f
R = Ph
O
CHMOAcineto cells
O2
R = 3-MeC6H4
R = 4-MeC6H4
R = 3-FC6H4
R = 4-FC6H4
R = 4-CH3OC6H4

R
R
(-)-2 or (+)-2
1
Scheme 1 Biotransformation of various 4-substituted cyclohexanones by CHMOAcineto
.
In our study, 4-phenylcyclohexanone (1a) was chosen as a
model substrate. Reversing the enantiopreference of
CHMOAcineto is challenging, since this enzyme is highly selective
in the BV oxidation of various substrates (Table S2, ESI†).8 To
reduce experimental screening efforts, the rational design of
novel CHMOAcineto catalysts with reversed enantiopreference
was guided by structure-based simulations. The crystal
structure of CHMOAcineto is not available, so we built a homology
model (named as CHMOhomo) based on the crystal structure of
CHMO from Rhodococcus sp. strain HI-31 (PDB code: 4RG3)12,
which exhibits high sequence similarity (Fig. S1, ESI†) around
the active sites with CHMOAcineto. To probe the key amino acid
residues that may affect the enzyme's enantioselectivity,
molecular docking and 20-ns molecular dynamics (MD)
simulation were performed on the WT CHMOhomo (Fig. 1A, Fig.
S4 and Fig. S5A, ESI†). Since the product should be also
compatible in the binding pocket of the enzyme and reflect the
enantiopreference of the enzyme, product 2a was used as the
model ligand in the simulations. It should be noted that under
the catalysis of WT CHMOAcineto, the product 2a with (S)-
configuration13a (98% ee (-), entry 1 in Table S2, ESI†) was
obtained. Analysis of the binding mode of 2a in WT disclosed
that (S)-2a is stabilized in the binding pocket, and the equatorial
phenyl-substituent of (S)-2a forms a π-π stacking with the side
chain of F432, contributing to the preference of an equatorial
phenyl group (Fig. 1A, Fig. S5A, ESI†). In contrast, (R)-2a could
not be docked into the active pocket (Fig. S4B, ESI†), which is in
line with the experimental data (Table S2, ESI†).
Fig. 1 MD optimized structure of WT CHMOhomo in complex with (-)-2a (green) (A), and
mutant F432L in complex with (+)-2a (salmon) (B), and mutant L435A in complex with
(+)-2a (salmon) (C). WT residues (F432 and L435) are highlighted in orange and the
mutation F432L in (B) and L435A in (C) in red. The other residues are shown in surface:
L143-yellow, P431-cyan, T433-lightblue, N434-slate, W490-lightgray, F505-wheat.
Cofactors (FADHOO- and NADP+) are represented by sticks in white.
target residues and reduce the screening efforts, we initially
mutated these sites into the amino acids, which have
significantly different sizes from the inherent one, and
therefore may reshape the binding pocket substantially. Thus
the large-sized F432 and F505 residues lining the top binding
side of the enzyme were mutated into a very small and medium
nonpolar amino acid (Alanine and Leucine); Likewise, the
medium-sized leucine at positions 143 and 435 lining the
bottom binding side was changed into small alanine and large
phenylalanine, respectively. Strikingly, complete reversal in
favor of (R)-2a (95% ee (+) in Table 1 and 98% ee (+) in Table 2)
was achieved by F432L and L435A. However, the mutantions at
position L143 and F505 did not show any effect on the
enantiopreference (Table S3, ESI†). These initial experimental
results implied that F432 and L435 are essential for the reversal
of enantiopreference. Moreover, MD results also showed that
the Ph-group of (S)-2a was closer to the residues F432 and L435
than F505 and L143 (Fig. S5A, ESI†). To gain a deep
understanding on the effect of the key residues F432 and L435,
two saturation mutagenesis libraries at F432 and L435 were
constructed and tested.
The mutants F432X were first examined toward ketone 1a.
The results revealed that similar to F432L, F432I showed the
preference for (R)-2a (85% ee (+), entry 1 in Table 1). However,
all other mutants of F432 preserved (S)-enantioselectivity,
similar to WT (Table S4, ESI†). These results indicated that (R)-
selectivity in the model reaction has strict spacial requirement
for the residue at position 432. When F432 is changed from G/A,
to V, and further to L/I, the volume of side chains increases
accordingly. However, only L/I amino acids are favourable for
the (R)-configuration enantiomer. Obviously, other mutations
with larger side chain than L/I (such as F432Y, F432W, F432P
etc.) are not suitable for the reversed selectivity (Table S4, ESI†).
Previous QM/MM calculations on the oxygenation of
cyclohexanone,
4-methylcyclohexanone14a
and
4-
hydroxycyclohexanone 14b indicated that the pose of substrates
in the binding pocket and the preferred direction of the
fragmentation of Criegee intermediate were critical for the
enzyme's enantioselectivity (Fig. S2, ESI†). The well-defined
position of ε-caprolactone product (Fig. S3, ESI†) in the active
sites featuring a certain directionality of the insertion of an
oxygen atom was indicated by theoretical investigation14a
,
which was in line with the crystal pose of ε-caprolactone in the
enzyme (PDB code: 4RG3)12. Due to the similarity in the reaction
mechanism and optimized geometry, 4-bulky substituted-
cyclohexanones were subjected to BV oxidation, similar to
unsubstituted cyclohexanone, thus the preferred orientation
(equatorial or axial) of their substituent group would ultimately
decide the binding mode of the substrates, resulting in different
stereoselectivity. Therefore, we propose the residues L143,
F432, L435, F505 located above or below the 4-Ph group in 2a
have the potential to affect the enantioselectivity (Fig. 1A).
To make a preliminary assessment of the importance of these
2 | J. Name., 2012, 00, 1-3
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Table 1 The mutants F432L and F432I as the catalysts in the desymmetrization of
prochiral cyclohexanones 1a
range from 51% to 78%, still maintaining high enantioselectivity
(>95% ee) (Table S6, ESI†). The present results demonstate the
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DOI: 10.1039/C8CC09951K
synthetic utility of these mutants.
F432L
Conv. (%)b
F432I
Conv. (%)b
To gain insight into the source of notably reversed
enantiopreference, molecular simulations were also performed
for mutants F432L and L435A (Fig. 1 and Fig. S5, ESI†). Based on
the preferred geometry proposed by theoretical
investigations14, the docked poses of (S)-2a shown in the active
pocket of F432L and L435A are unproductive because of the
unfavorable insertion of oxygen atom (Fig. S4C and S4E, ESI†),
while (R)-2a is located preferably in the reshaped active pocket
(Fig. S4D and S4F, ESI†). Interestingly, MD result in Fig. 1B
showed that the mutation F432L causes the adjacent loop to
undergo a significant conformational change. Accordingly, the
side-chain of L432 on the loop is rotated inward the active
pocket so that the “top” space of the binding pocket was
reduced. As a result, the side-chain of L432 impeded the
potential orientation of the equatorial substituent, thus the
unproductive pose (S)-2a with the equatorial substituent
rotated 180° through the main axis (Fig. S4C, ESI†). Moreover,
the reduced space due to the mutation may fasten the axial
substituent of (R)-2a in the binding pocket by forming CH-π
hydrogen bonds15 between the 4-Ph group and the side chain of
L143 and L435 (Fig. S5B, ESI†). Similarly, the extra space vacated
by mutating L435 into A or G is favourable to accommodate the
bulky axial substituent of (R)-2a, and the hydrophobic cavity
environment surrounded by L143 and A435 further stabilizes
the phenyl-substituent in axial orientation (Fig. 1C, Fig. S5C,
ESI†). Therefore, the simulation results account for the reversed
enantiopreference of both mutants F432L and L435A at atomic
level. When F432 and L435 are mutated into appropriate
residues, these residues lining the top and bottom of the active
pocket may serve as a “top” switch and a “bottom” switch, to
reverse the enantiopreference of the enzyme for the
sandwiched substrates.
Next, the substrate scope of the best single-point mutants
(F432L, F432I, L435A, L435G) engineered for substrate 1a was
studied. WT enzyme transformed all substrates 1b-1k into the
corresponding (-)- or (S)-products with the exception of 1h, for
which slight (+)-enantioselectivity is observed (33% ee) (Table
S2, ESI†). Notably, the four aforementioned best mutants
catalyzed the BV oxidation of the phenyl-derived
cyclohexanones 1b-1g, with good yields and highly reversed (+)-
enantioselectivities (up to 99% ee, and 99% conversion) (Table
1, 2), implying these mutants are indeed excellent catalysts with
the capability to reverse the enantiopreference of CHMOAcineto
with broad substrate scope. For the compounds 1j and 1k with
short-alkyl substituents, the mutants F432I and F432L provided
(S)-lactones with excellent ee, similar to WT (entries 10-11 in
Table 1). Interestingly, for all these four mutants, the degree of
reversed enantiopreference from (-)- to (+)-configuration
toward the alkyl substituted ketones correlates well with the
chain length of alkyl substituents. For example, the F432L
variant catalyzed the transformation of compounds 1j→1i→1h
that have the increased chain length, providing the products
with ee values of 98%(-)→77%(-)→85%(+) (Table 1, entries 10,
9, 8). L435G variant displays similar features for compounds
Entry Substrate
eep(%)c
eep(%)c
1
2
1a
1b
1c
1d
1e
1f
99
99
99
99
99
99
99
99
99
99
99
95(+)
95(+)
99
99
99
99
99
99
99
99
99
99
99
85(+)
95(+)
3
98(+)
97(+)
4
98(+)
95(+)
5
99(+)
92(+)
6
98(+)
98(+)
7
1g
1h
1i
97(+)
96(+)
8
85(+)
82(+)
9
77(S)(-)d
98(S)(-)d
99(S)(-)d
81(S)(-)d
98(S)(-)d
99(S)(-) d
10
11
1j
1k
a The whole cell experiments are described in Experiment section. b, c Determined
c
by chiral GC. The data of optical rotation was listed in ESI. dThe absolute
configuration was confirmed by comparison with the literature13b
.
Table 2 The mutants L435A and L435G as the catalysts in the desymmetrization of
prochiral cyclohexanones 1a
L435A
Conv. (%)b
L435G
Conv. (%)b
Entry Substrate
eep(%)c
eep(%)c
1
2
1a
1b
1c
1d
1e
1f
99
92
91
99
99
88
99
99
99
99
99
98(+)
84(+)
99
95
90
99
99
85
99
99
99
99
99
99(+)
91(+)
3
94(+)
96(+)
4
99(+)
99(+)
5
99(+)
99(+)
6
99(+)
95(+)
7
1g
1h
1i
85(+)
90(+)
8
30(-)
35(+)
9
60(R)(+)d
31(S)(-)d
62(S)(-)d
70(R)(+)d
28(S)(-)d
70(S)(-)d
10
11
1j
1k
a The whole cell experiments are described in Experiment section. b, c Determined
c
by chiral GC. The data of optical rotation was listed in ESI. dThe absolute
configuration was confirmed by comparison with the literature13b
.
It should be noted that F432S variant does not show reversed
preference for 1a, (Table S4, entry 12, ESI†), although it showed
reversed stereoselectivity for 4-hydroxycyclohexanone8a.
Further, L435 was mutated into 18 different amino acids,
complete reversal in favor of (R)-2a was achieved by L435A/G
variants with small side chains (entry 1 in Table 2). The mutants
with side chains longer than leucine displayed low catalytic
activity, whereas the rest mutants such as L435C, L435I, L435V
and L435T, still preserved the (S)-selectivity (Table S5, ESI†).
Considering the potential application, we further tested the
best mutants (F432L and L435A) and WT in a 2-gram-scale
conversion of 1a with a final concentration 12mM in 1L reaction
system. The final conversion (95%-WT, 92%-F432L, 70%-L435A)
was achieved within 36h, and the isolation yields are in the
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Dong, E. Fernández-Fueyo, F. Hollmann, C. E. Paul, M. Pesic, S.
1k→1j→1i (70%(-)→28%(-)→70%(+), entries 22, 21, 20 in Table
2). The long alkyl substituent is perferred for (+)-selectivity in BV
oxidation reactions catalyzed by these mutants. Except for 4-n-
propyl substituted cyclohexanone (1i) (Table 2, entry 9), other
ketones with short alkyl substitutents at C-4 position(1h, 1j, and
1k), or 2-methyl cyclohexanone (3) and 3-methyl
cyclohexanone (6) (Table S7, ESI†) can not be transformed with
reversed selectivity, which outlines the limitation of these
mutants discussed herein. This is probably because of the
mobility of the smaller substrate in the reshaped active site of
these mutants. For example, in the case of the “bottom” switch
(Fig. 1C, Table 2), the extra space vacated by mutations of L435A
or L435G is favorable for the orientation of the axial substituent,
especially for long axial-substituent which may bind more
tightly with the reshaped pocket to match the axial space
created by the mutation. However, the short substituent of the
substrate (1j, 1k) may prefer to staying in equatorial rather than
in axialdue to lack of potential binding with the enlarged pocket
along the axial, such that (S)-selectivity would be still preserved.
In conclusion, starting from WT CHMOAcineto, we found a “top”
or “bottom” switch at position 432 and 435, which can reverse
the enantiopreference toward bulky substituted-ketones
sandwiched between the two switches. By single mutation at
either position 432 or 435, complete reversal of
enantiopreference has been achieved. The rational approach
combining in silico and experimental studies supersedes the
random selection of target residues for mutagenesis, and
therefore has drastically reduced the screening effort.
Sequences alignment based on the 94 BVMO sequences (Fig. S7,
ESI†) implied these two switch residues are highly conserved in
many BVMO, thus the finding herein will provide a valuable
guidance for facile engineering other BVMO with reversed
enantiopreference for specific substrates.
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This work was supported by National Natural Science
Foundation of China (21574113, 21472169) and Zhejiang
Provincial Natural Science Foundation (LY19B020014). The
authors thank the computing resource from the high
performance computing cluster at Queen’s University Belfast.
Conflicts of interest
There are no conflicts to declare.
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Table of Contents
“Top” or “Bottom” Switches of a Cyclohexanone Monooxygenase Controlling
the Enantioselectivity of the Sandwiched Substrate
Yujing Hu,a, b Jie Wang,a Yixin Cen,a He Zheng,a Meilan Huang,* b Xianfu Lin,a Qi
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Single mutation F432I/L or L435A/G remarkably reversed the (-)-selectivity of WT CHMOAcineto
.