Directed Evolution of an Artificial Imine Reductase

Article abstract of DOI:10.1002/anie.201711016

Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin-streptavidin technology using a straightforward procedure allowing screening in cell-free extracts. Two streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines. The evolved ATHases were stable under biphasic catalytic conditions. The X-ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor, thus increasing exposure of the metal to the protein surface and leading to a reversal of enantioselectivity. This hypothesis was confirmed by a multiscale approach based mostly on molecular dynamics and protein–ligand dockings.

Full text of DOI:10.1002/anie.201711016

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Accepted Article
Title: Directed Evolution of Artificial Imine Reductase
Authors: Martina Hestericová, Tillmann Heinisch, Lur Alonso-Cotchico,
Jean-Didier Maréchal, Pietro Vidossich, and Thomas R. Ward
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711016
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Angewandte Chemie International Edition
10.1002/anie.201711016
COMMUNICATION
Directed Evolution of Artificial Imine Reductase
[
a]
[a]
[b]
[b]
[b]
Martina Hestericová , Tillman Heinisch , Lur Alonso-Cotchico , J.-D. Maréchal* , Pietro Vidossich ,
and Thomas R. Ward*[a]
Abstract: Artificial metalloenzymes, resulting from incorporation
of a metal cofactor within a host protein, have received increasing
attention in the last decade. Herein, we report on the directed
evolution of an Artificial Transfer Hydrogenase (ATHase) based
on the biotin-streptavidin technology using a straightforward
protocol allowing screening in cell free extracts. Our efforts
yielded two streptavidin isoforms with improved catalytic activity
and selectivity for the reduction of cyclic imines. Gratifyingly, the
evolved ATHases proved stable under biphasic catalytic
conditions. The X-ray structure analysis reveals that introducing
bulky residues within the active site results in flexibility changes
of the cofactor, thus increasing exposure of the metal to the
protein surface and leading to a reversal of enantioselectivity. This
hypothesis was confirmed by a multiscale approach based mostly
on molecular dynamics and protein-ligand dockings.
Figure 1. Close-up view of the active site of an artificial imine reductase. The protein
is displayed as solvent accessible surface and the biotinylated cofactor as color-coded
stick. Positions selected for mutagenesis are highlighted (PDB 3PK2).
Biocatalysis offers an attractive means to produce high-
added value products.[1] Its limitations in reaction repertoire,
substrate scope and operational stability can be overcome using
These combine attractive features of enzymatic catalysis with the
directed evolution or encapsulation techniques.[2]
vast reaction repertoire of organometallic catalysis.[13] In this
Enantiopure amines, representing important intermediates
context, the biotin-streptavidin technology[14] has proven versatile,
for synthesis of biologically active compounds, agrochemicals,
allowing the development of numerous ArMs that have been
and flavors, are gaining increasing importance in both academic
and industrial sectors. Indeed, approximately 40 % of all
optimized to catalyze new-to-nature transformations.[15]
In order to fine-tune the performance of enzymes, directed
pharmaceuticals contain at least one enantiopure amine moiety.[3]
evolution has proven extremely powerful.[16] Consisting of iterative
There is thus a high demand for versatile tools to produce these.
cycles of (random) mutagenesis, protein overexpression and
Current strategies include: homogeneous catalysis and organic
screening, this versatile technique allows to incrementally
synthesis,[4] resolution of racemates or biocatalysis.[6] Amine
[5]
improve a targeted feature of an enzyme: activity, selectivity,
[
7]
[8]
dehydrogenases,
phenylalanine
ammonia
lyases,
stability etc. As ArMs consist of an abiotic cofactor and a
genetically-encoded host protein, directed evolution can also be
applied to the optimization of such hybrid catalysts.[
transaminases[ and imine reductases have been reported for
the synthesis of enantiopure amines. The dynamic kinetic
resolution of amines can be achieved using monoamine
oxidases[ or lipases . Following the first reports by Mitsukura
in 2010,[10d] imine reductases have gained significant interest from
the synthetic community. The limited number of available imine
9]
[10]
17]
To speed-up the directed evolution of ArMs, we set out to
perform catalysis using E. coli cell free extracts (cfe) rather than
purified protein samples. Previously, we identified glutathione as
a main contaminant,[18] leading to the irreversible poisoning of the
precious metal cofactor when catalysis is performed in cfe.
Addition of diamide (1,1-azobis(N,N-dimethylformamide, DiAm) to
cfe containing streptavidin (Sav) prior to the addition of the
cofactor significantly restores the catalytic performance of
ArMs.[ To test the usefulness of this screening protocol, we set
out to optimize the performance of an artificial imine reductase
based on the biotin-Sav technology by directed evolution.
In this work, we present the implementation of a streamlined
optimization protocol,[19] which allows us to screen protein
variants using cfe pre-incubated with DiAm. Upon screening 300
variants contained in cfe, which corresponds to four weeks of
mutagenesis and expression, four weeks of protein purification,
and 200 hours of analytical measurements, two mutants with an
increased catalytic activity and opposite enantioselectivity for the
reduction of cyclic imines were identified.
11]
[12]
[
10]
reductases calls for alternative biocatalytic approaches.
In the past fifteen years, biocatalysis has been
complemented with artificial metalloenzymes (ArMs).
18b]
[
[
a]
b]
M. Hestericová, Dr. T. Heinisch, Prof. Dr. T. R. Ward
Department Chemistry
University of Basel
Mattenstrasse 24a, BPR 1096, Basel, 4002 (Switzerland)
E-mail: Thomas.ward@unibas.ch
Lur Alonso-Cotchico, Dr. Pietro Vidossich, Prof. Dr. Jean-Didier
Maréchal
Departament de Química
Universitat Autònoma de Barcelona
Edifici C.n., 08193 Cerdonyola del Vallès, Barcelona (Spain)
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
To improve the catalytic performance of the ATHase, we
selected a volume of 10 Å around the iridium center anchored
within Sav and subjected the corresponding amino acids to
iterative saturation mutagenesis (Figure 1). As starting point for
the screen, we selected SavK121A. For the first generation of the
directed evolution, a reduced library of amino acids was included.
At positions T111, S112, G113, T114, A116, N118, S122, T123
and L124, the following residues were introduced relying on
precise primers: A, V, L, D, E, Q, K, H, M, Y, S, P (or N if one of
the targeted mutation is present at this position). For the following
generations, all canonical amino acids were individually
introduced. Mutants were overexpressed in E. coli in 96 deep-well
plates, and the cells were lysed, incubated with DiAm for 15
minutes, supplemented with [Cp*Ir(biot-p-L)Cl] and screened for
the transfer hydrogenation of cyclic imines 1a, 1b and 1c
(
Scheme 1, See SI for details). After identification of improved
variants using cfe, the corresponding mutants were
overexpressed in 1 L autoinduction medium,[20] purified by
iminobiotin-sepharose affinity chromatography and lyophilized.
The pure Sav mutants were then tested for their ATHase activity.
In the first generation, positions S112 and N118 were
screened, yielding (S)- and (R)-selective mutants K121A-S112R
and K121A-N118P (Table 1, entry 4 and 5; Figure 2). Next,
K121A-N118P was subjected to focused saturation mutagenesis
at position S112. Mutant K121A-N118P-S112A yielded (R)-2a
with 86 % ee (entry 6). Position S122 was mutated next.
Substituting the polar serine to a methionine further improved the
enantioselectivity for the reduction of 1a, yielding (R)-2a in 92 %
ee and full conversion (conv., entry 7). We hypothesized that
mutating alanine to bulky arginine at position 112 would afford the
opposite enantiomer of 2a. To our delight, the resulting Sav
variant produced (S)-2a in 63 % ee and with improved conv. (entry
Figure 2. Summary of the directed evolution path to afford both an (R)- and an
(S)-selective ATHase for the reduction of 1a.
In stark contrast, [Cp*Ir(biot-p-L)Cl] · K121A proved less active
under biphasic conditions (entry 3). Preparative-scale
experiments (>120 mg of 1a) resulted in >99 % HPLC yield (70 %
isolated yield) and 91 % ee (R) for S112A-N118P-K121A-S122M
and >75 % HPLC yield (55 % isolated yield) and 71 % ee (S) for
S112R-N118P-K121A-S122M-L124Y (Table 1, entry 9 and 13).
The resulting Sav library was tested for the reduction of the
bulky isoquinoline 1b as well as cyclic imine 1c (Table S3). For
the reduction of 1b, an (S)-selective ATHase was identified;
S112T-N118K-K121A-S122K affords (S)-2b in 50% ee and 75 %
conv. (Table S3, entry 8). The highest (S)-selectivity for the
reduction of 1c was obtained with S112R-N118P-K121A-S122M-
L124Y, yielding (S)-2c in 55 % ee and 86 % conv. (Table S3, entry
22).
9). The (S)-selectivity was further improved by introducing
tyrosine instead of lysine at position 124, which yielded (S)-2a
with 78 % ee and full conv. (entry 10).
Because of the low solubility of 1a in water, we performed
experiments under a biphasic set-up[21] with 100 mM 1a dissolved
in ethyl acetate. To our delight, improved enantioselectivity was
observed for both (R)- and (S)-selective ATHases (entry 8 and
Table 1. Selected results for the reduction of cyclic imine 1a using purified
proteins.[
a]
ee
conv.
(%)[d]
Entry
Sav
TON
12).
(%)[d]
1
2
3
4
5
6
7
8
9
[Cp*Ir(biot-p-L)Cl]
K121A
0
17
99
35
54
31
59
-27
86
92
95
91
-63
-78
198
100
193
72
K121A [b]
5
N118P-K121A
96
S112R-K121A
36
S112A-N118P-K121A
S112A-N118P-K121A-S122M
S112A-N118P-K121A-S122M[b]
S112R-N118P-K121A-S122M[c]
S112R-N118P-K121A-S122M
S112R-N118P-K121A-S122M-L124Y
S112R-N118P-K121A-S122M-
L124Y[b]
100
100
19
200
200
380
198
179
198
99 (70e)
1
0
1
90
1
99
1
2
3
-85
-72
11
220
150
S112R-N118P-K121A-S122M-
L124Y[c]
1
75 (55e)
[
[
a]
b]
The reactions were performed with 10 mM substrate at 37°C for 48 h (see SI).
Reactions were performed in a biphasic system with 100 mM substrate at RT
[
c]
for 4 days.
Preparative-scale reaction, 124 mg of 1a were added to the
reaction mixture (60 ml). [ Enantiomeric excess and conv. were determined by
d]
HPLC. Positive ee values correspond to (R)-product and negative ee values
correspond to (S)-product. Isolated yield.
Scheme 1. Reduction of cyclic imines 1a, 1b and 1c using ATHase based on
the biotin-streptavidin technology.
[e]
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COMMUNICATION
within the Sav monomer-monomer interface results in a nearly
1
S
80° rotation of the piano stool moiety around the Cbenzene
sulfonamide bond. The position of the pianostool is stabilized by an
H-bond between the Y124 hydroxyl and the cofactor amino group
Figure S7b,c). Unfortunately, no electron density was present to
-
(
model the {Cp*IrCl} moiety. We speculate that is may be due to
increased flexibility of the surface-exposed complex or partial
dissociation of the {Cp*IrCl}. Increased cofactor exposure
presumably leads to increased solvent exposure of the substrate
(
Figure S7b), reminiscent to the structure of [Cp*Ir(biot-p-L)Cl] ·
S112K (PDB 4OKA).[ Importantly, both crystal structures of the
evolved ATHases reveal increased atomic B-factors within the
loop-7,8 and the surface-exposed terminus of the cofactor when
compared to published ATHases S112A and S112K (PDB 3PK2
and 4OKA) (Figure S10, Table S5).[18] In the latter structures, the
loop-7,8 is conformationally rigidified by an H-bonding network
including i) the N118 side chain amide, ii) a water molecule and
iii) the T115 backbone amide. Mutation N118P results in
elimination of this H-bonding network (Figure S11a). Additional
flexibility is produced in loop 7,8 through mutation S122M that
induces breaking of H-bonds in a Sav monomer-monomer
interface (Figure S11b). These observations are reminiscent of
22]
Scheme 2.
- Saturation kinetic data for selected ATHases based on
incorporation of [Cp*Ir(biot-p-L)Cl] (2 eq.) in various Sav isoforms (1 eq.) for the
reduction of imine 1a or 1b. Error bars represent ± standard deviation.
Next, the saturation kinetic behavior of the bare cofactor and
the best performing ATHases was determined (Scheme 2; Table
S4). Variant K121A displays an 8-fold increased kcat value for the
reduction of 1b compared to the bare cofactor. Moreover, both
mutants S112A-N118P-K121A-S122M and S112R-N118P-
K121A-S122M-L124Y display improved reaction rates for the
reduction of 1b compared to the bare cofactor. Introducing a bulky
the elevated atomic B-factors found in the crystal structure of a
Sav-based metathase.[17f]
Crystal structures were determined assuming saturation of
Sav monomers by the cofactor. However, catalysis was
performed under a 1:2 ratio of cofactor per Sav monomer. To
structurally characterize the assemblies under catalytic conditions,
Molecular Dynamics (MD) simulations were performed using an
implicit solvent approach and applying metadynamics to initially
boost the exploration of conformational space (see SI). The
tyrosine residue at position 124 has a dramatic effect on K
increasing its value to 50 mM from 7.4 mM for the bare cofactor.
Both tested ATHases display very similar K values, while their
cat differ significantly. This suggests that introducing a second
M
,
M
k
bulky substituent contributes to stabilization of the reaction
transition state, thus improving the reaction rate for the (S)-
selective ATHase. Substrate inhibition is encountered for mutants
K121A and S112R-N118P-K121A-S122M-L124Y, whereas the
free cofactor and mutant S112A-N118P-K121A-S122M display
classical Michaelis–Menten kinetics. Unlike the results obtained
with 1b, the reduction of 1a by ATHases revealed that none of the
mutants could reach substrate inhibition due to limited solubility of
-
achiral planar [Ir(III)] 16e two-legged pianostool was considered.
Conformations from the MD trajectories were grouped (clustered)
based on the cofactor position in the vestibule. The number of
configurations in each group (cluster) is displayed in Figure
S13a,b together with an estimate of the interaction energy
between the [Ir] complex excluding the biotin fragment and the
protein (Figure S13c,d). Variant S112R-N118P-K121A-S122M-
L124Y was computed to bind the cofactor more tightly than variant
S112A-N118P-K121A-S122M. This observation may be
explained based on the polarity of the residues in the vestibule,
which is more hydrophobic for S112A-N118P-K121A-S122M.
1a. Compared to the bare cofactor, all mutants display slightly
higher kcat coupled with lower K values.
M
To gain structural insight into the best performing ATHases,
crystals of the most evolved variants were soaked with an excess
of [Cp*Ir(biot-p-L)Cl]. Inspection of the X-ray structure of
[
Cp*Ir(biot-p-L)Cl] · S112A-N118P-K121A-S122M (Figure 3a)
highlights the structural similarities to the structure of [Cp*Ir(biot-
p-L)Cl] · S112A.[22] The overall ATHase structure and the position
and absolute configuration of the piano stool moiety are virtually
identical (RMSD of all C = 0.693). Due to potential steric clashes
between two symmetry-related cofactors, the chloride ligand was
not modeled. However, the crystal structure suggests the
preferred formation of an (S)-configuration at the metal for
[
Cp*Ir(biot-p-L)Cl] (i.e. (R)-configuration for the catalytically active
hydride [Cp*Ir(biot-p-L)H]) (Figure S6a). The piano stool
localization is stabilized by an H-bond between the A121
backbone carbonyl oxygen and the piano stool amine nitrogen.
As the cationic lysine at position 121 is substituted by an apolar
alanine, it prevents a possible interaction with the imine nitrogen
of the substrate. The non-concerted transition state allowing
CH⋅⋅⋅π interaction between the Cp* moiety and the phenyl of 1a
allows for the formation of (R)-2a. The X-ray structure of
Figure 3. Close-up view of the crystal structure of [(Cp*)Ir(Biot-p-L)Cl] · S112A-
N118P-K121A-S122M (PDB 6ESS) (A) and [(Cp*)Ir(Biot-p-L)Cl] · S112R-
N118P-K121A-S122M-L124Y (PDB 6ESU) (B). The protein is displayed as
transparent surface and cartoon model with mutations highlighted in red. For
clarity, one cofactor per Sav tetramer is displayed. The cofactor is contoured
with electron density from a 2FoFc map in marine (1.0 ) and an anomalous
dispersion density map in red (4.0 ). Magenta atoms are not resolved in the
electron density.
[
Cp*Ir(biot-p-L)Cl] · S112R-N118P-K121A-S122M-L124Y (Figure
b) reveals that introducing bulky residues S112R and L124Y
3
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COMMUNICATION
Accordingly, the MD trajectory of S112R-N118P-K121A-S122M-
L124Y shows reduced mobility of the cofactor compared to
S112A-N118P-K121A-S122M. The conformation of the cofactor
in the most populated cluster in the trajectory of S112A-N118P-
K121A-S122M is very similar to the X-ray determination (Figure
S14a). However, further conformations are accessible to the
cofactor via rotation around the S–N bond (Figure S14b). Similarly,
the cofactor conformation in the most populated cluster in the
trajectory of S112R-N118P-K121A-S122M-L124Y is consistent
with the X-ray structure (Figure S12c).
Next, we used this structural insight to rationalize the
opposite enantioselectivity resulting from the most evolved
ATHases towards substrate 1a. For this purpose, we docked the
protonated form of 1a to the representative structures of selected
clusters. The resulting structures were energy-minimized and the
substrate binding energy estimated. We did not attempt to
estimate reaction energies (i.e. with QM/MM methods).
In the most populated cluster from the MD trajectory of
S112R-N118P-K121A-S122M-L124Y, [Cp*Ir(biot-p-L)H] leads to
the formation of (R)-2a. The best docking solutions for the pro-R
and pro-S faces of 1a project the phenyl substituent in a pocket
formed by residues R112 and Y124 from both monomers and
P118 of the adjacent monomer. The difference between the two
binding poses consists in the orientation of the imine plane: the
NH group points towards the hydroxyl of tyrosine Y124 in the pro-
S structure (Figure 4a), whereas it is rotated by 180 degrees and
does not display interactions in the pro-R structure (Figure 4b).
From a docking point of view, the pro-R binding mode is slightly
favored over the pro-S one. However, MD simulations starting
from these structures reveal that the pro-R binding mode is not
stable, whereas the pro-S displays a longer lifetime (i.e. about 200
ps). During this time, the NH group reorients to form an H-bond to
the backbone carbonyl of A121. Therefore, we conclude that
Figure 4. Substrate 1a docked to the most populated cluster from MD
simulations of variants S112R-N118P-K121A-S122M-L124Y (panel a and b)
and S112A-N118P-K121A-S122M (c). The cofactor is shown in blue stick, with
the Cp* group in cyan and the Ir-H atoms as balls; the substrate is shown as
sticks; the protein surface is shown in gray, with the residues within 4 Å colored
by type: white for hydrophobic residues, green for polar, blue for positively
charged.
Acknowledgements
This research was supported by the Swiss Nanoscience Institute,
and advanced ERC grant (DrEAM) and the SNF (grant 200020
2 2
162348). TRW thanks Umicore for a generous loan of [Cp*IrCl ] .
JDM, PV and LAC are thankful for the support given by the
Spanish grant CTQ2014-54071-P, Generalitat de Catalunya grant
2014SGR989 and the COST Action CM1306.
[
Cp*Ir(biot-p-L)H] · S112R-N118P-K121A-S122M-L124Y should
preferentially reduce the pro-S face of 1a, an observation in
agreement with the experiments.
Concerning mutant S112A-N118P-K121A-S122M, docking of 1a
to the representative structure of the most populated cluster
Keywords: artificial metalloenzymes • directed evolution •
enzyme catalysis • transfer hydrogenation • cyclic imines
(
which would form the (R)-configuration of the metal-hydride)
results in a binding mode with no clear preference for the
reduction of either the pro-R or the pro-S faces of the substrate
[
1]
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1
a. In this binding mode, 1a sits on the hydrophobic surface
formed by residues L110, A112, T114, P118, A121, L124 (Figure
c). We also considered binding of 1a to representative structures
8973.
4
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2]
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most populated. The MD simulations started from these bound
structures did not reveal any alternative long-lived binding mode.
It seems then that the ee observed experimentally for this mutant
cannot be easily rationalized from a pure binding process and
further aspects including the calculation of reaction barriers may
be required.
In summary, introduction of [Cp*Ir(Biot-p-L)Cl] within Sav
isoforms affords ATHases. These can be optimized by directed
evolution protocols using unpurified cfe, thus dramatically
improving the throughput of the effort. Two mutants with
increased reaction rates and enantioselectivities were identified
for the reduction of cyclic imine 1a, allowing for the formation of
both (R)-2a (95 % ee) and (S)-2a (86 % ee). The possibility of
performing catalysis in a biphasic medium paves the way for
greater scope of applications of such hybrid catalysts.
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Angewandte Chemie International Edition
10.1002/anie.201711016
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Entry for the Table of Contents
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Martina Hestericová, Tillman Heinisch,
Lur Alonso-Cotchico, J.-D. Maréchal*,
Pietro Vidossich, and Thomas R. Ward*
Page No. – Page No.
Directed Evolution of Artificial Imine
Reductase
Skipping steps: An artificial imine reductase is generated by incorporation of a
biotinylated iridium pianostool complex within streptavidin. Application of an
streamlined screening protocol allows for a faster screening in cell free extracts. With
this system, both enantiomers of product in high ee can be obtained after only several
rounds of saturation mutagenesis.
This article is protected by copyright. All rights reserved.