Simultaneous engineering of an enzyme's entrance tunnel and active site: The case of monoamine oxidase MAO-N

Article abstract of DOI:10.1039/c6sc05381e

A new directed evolution approach is presented to enhance the activity of an enzyme and to manipulate stereoselectivity by focusing iterative saturation mutagenesis (ISM) simultaneously on residues lining the entrance tunnel and the binding pocket. This combined mutagenesis strategy was applied successfully to the monoamine oxidase from Aspergillus Niger (MAO-N) in the reaction of sterically demanding substrates which are of interest in the synthesis of chiral pharmaceuticals based on the benzo-piperidine scaffold. Reversal of enantioselectivity of Turner-type deracemization was achieved in the synthesis of (S)-1,2,3,4-tetrahydro-1-methyl-isoquinoline, (S)-1,2,3,4-tetrahydro-1-ethylisoquinoline and (S)-1,2,3,4-tetrahydro-1-isopropylisoquinoline. Extensive molecular dynamics simulations indicate that the altered catalytic profile is due to increased hydrophobicity of the entrance tunnel acting in concert with the altered shape of the binding pocket.

Full text of DOI:10.1039/c6sc05381e

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Simultaneous engineering of an enzyme’s entrance tunnel and
active site: The case of monoamine oxidase MAO-N
Received 00th January 20xx,
Accepted 00th January 20xx
Guangyue Lia, b,§, Peiyuan Yaoc, §, Rui Gongc,§, Jinlong Lic, Pi Liuc， Richard Lonsdalea, b, Qiaqing Wuc,
Jianping Lin c,*，Dunming Zhuc,* and Manfred T. Reetza, b,
*
DOI: 10.1039/x0xx00000x
A new directed evolution approach is presented to enhance the activity of an enzyme and to manipulate
stereoselectivity by focusing iterative saturation mutagenesis (ISM) simultaneously on residues lining the
tunnel and the binding pocket. This combined mutagenesis strategy was applied successfully to the
monoamine oxidase from Aspergillus niger (MAO-N) in the reaction of sterically demanding substrates which
are of interest in the synthesis of chiral pharmaceuticals based on the benzo-piperidine scaffold. Reversal of
enantioselectivity of Turner-type deracemization was achieved in the synthesis of (S)-1,2,3,4-tetrahydro-1-
www.rsc.org/
methyl-isoquinoline,
(S)-1,2,3,4-tetrahydro-1-ethylisoquinoline
and
(S)-1,2,3,4-tetrahydro-1-
isopropylisoquinoline. Extensive molecular dynamics simulations indicate that the altered catalytic profile is
due to increased hydrophobicity of the entrance tunnel acting in concert with the altered shape of the
binding pocket.
the tunnel even when X-ray data is available. In some cases, the
tunnel acts as a gate-keeper (molecular filter), which influences
Introduction
Directed evolution constitutes a versatile tool for engineering the
catalytic profiles of enzymes, stereoselectivity, activity and
substrate specificity being of particular interest in organic chemistry
and biotechnology.1 Since screening is the labor-intensive step
(bottleneck of directed evolution), efforts continue to focus on
developing efficient mutagenesis strategies which enable the
production of high-quality libraries requiring minimal analytical
work.1,2 Iterative saturation mutagenesis (ISM)3 at sites lining the
binding pocket is one of several approaches which deliver small and
“smart” mutant libraries. Accordingly, sites labeled A, B, C, D, etc.,
each comprising one or more residues, are first identified using X-
ray data or homology models, followed by the generation of mutant
libraries and respective screening. The best hit originating from one
library is then used as a template for randomization at another site.
In many enzymes the active site is buried deeply within the
core of the protein, which means that in order to access the binding
pocket, substrates must pass first through the protein via an
entrance tunnel.4 Unfortunately, sometimes it is difficult to identify
substrate selectivity and sometimes even activity as shown by
mutagenesis experiments.5 Interest in identifying and
understanding how these tunnels exert such control has grown
steadily over the past several years,4 leading to the realization that
in such cases tunnel optimization and binding pocket manipulation
are both necessary for optimal catalytic performance. However,
directed evolution studies encompassing mutagenesis at both types
of sites are rare. A recent example is due to Hauer, Pleiss and
coworkers, who performed separate saturation mutagenesis
experiments at the active site and the entrance tunnel of a P450
monooxygenase, and subsequently combined the respective
positive mutations.6
The purpose of the present study was to apply ISM with the
aim of manipulating the size and polarity of an enzyme’s entrance
tunnel as well as the shape of its binding pocket. In principle, this
can be attempted either sequentially or simultaneously. In the
present study we decided to test the latter option as a new strategy
in directed evolution. As will be seen, the substrate of interest is not
accepted by the wild-type (WT) enzyme, which may be due to two
different phenomena: The particular size and/or polarity of the
tunnel prevents smooth substrate entry and product release,
and/or the specific shape of the binding pocket is not conducive to
catalytic turnover. If both effects operate, then focusing on them
sequentially may fail to provide maximally improved variants.
In order to explore such a scenario, the monoamine oxidase
from Aspergillus niger (MAO-N)7 was chosen as the model enzyme.
In a series of previous studies by the Turner group devoted to the
efficient deracemization of primary,8 secondary9 and tertiary
amines10 as well as chiral heterocycles such as substituted
12
pyrrolidines and tetrahydro-isoquinolines,11,
this enzyme has
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already been subjected to directed evolution using mutator strain (green). B): Residues surrounding the substrate access tunnel (red)
technology as well as saturation mutagenesis at the binding pocket likewise chosen for mutagenesis (shown in green).
and separately at the putative entrance to the active site.12
access tunnel [W94, F119, F128, H142, E145, L213, M242, L245,
Although the crystal structure of wildtype (WT) MAO-N remains to
I246, T354, Y365 and I367] (Fig. 1).
be analyzed, X-ray structures of a 3-point mutant MAO-N-D3 and a
These two types of residues share four common residues
5-point variant MAO-N-D5 have been reported13 which helped in
[W94, L213, L245 and I246]. Consequently, we chose 23 residues for
the interpretation of part of the data. The total length and size of
potential mutagenesis and split them into six groups as displayed in
the entrance tunnel were not explicitly defined.12,13 It should be
Table 1. Saturation mutagenesis at a 4-residue site using NNK codon
pointed out that further protein engineering studies of MAO-N and
degeneracy encoding all 20 canonical amino acids would require for
of other MAOs have also appeared.14
95% library coverage excessive screening (3 x 106 transformants).2
Therefore, the reduced amino acid alphabet NDT was considered,
Results and discussion
encoding 12 amino acids (Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys,
As model compounds for assessing the activity of MAO-N mutants, Arg, Ser, Gly), which requires the screening of about 6 x 104
we chose amines 1,2,3,4-tetrahydro-1-methylisoquinoline (1), transformants for 95% library coverage.2 Although this is still a very
1,2,3,4-tetrahydro-1-phenylisoquinoline (2) and 2-phenyl-pyrroline high number, a previously developed easy-to-perform colorimetric
(3) as shown in Scheme 1. The plan was to improve the activity on-plate assay18 for rapidly identifying thousands of active variants
towards amines 1 and 2 and to control stereoselectivity, these was applied in all mutagenesis experiments. The most active
chiral compounds being of particular interest because the structural variants were subsequently tested as catalysts in Turner-type
motifs occur in therapeutic drugs such as the proton pump inhibitor deracemization reactions.
YH188515 and Solifenacin,16 respectively. In our hands the
compounds showed no activity in attempted WT MAO-N catalyzed for substrate 3 (Supporting Information Table S1), but only those in
reactions, in contrast to amine 3 which led to a slow but detectable library proved to be hits for substrates and 2, e.g.,
Libraries A, B, D, E and F contained a number of variants active
F
1
transformation (Supporting Information Scheme S1). Therefore, we W230R/W430C and W230I/T354S/W430R (Supporting Information
chose 3 as the model detector substrate for identifying functional Tables S2 and S3). Based on the result of sequence determination,
enzyme residues that can then be used in ISM experiments for we identified all activity related positions in sites A, B, D and E,
evolving variants that also accept substrates 1 and 2. Upon creating namely F128, L213, C214, M242, Y365 and I367 (Supporting
a homology model based on MAO-N-D3 (N336S/M348K/I246M), Information Table S1). Consequently, we regrouped these positions
the CAVER algorithm17 was applied with the aim of identifying the into sites G (F128, L213 and C214) and H (M242, Y365 and I367)
tunnel. In order to design an appropriate ISM scheme for MAO-N (Supporting Information Fig. S1-a). Thereafter, ISM was applied
engineering, we considered two types of residues: Amino acid using the best variants as templates. The results are summarized in
positions lining the substrate 1 in the binding pocket [T93, W94, the Supporting Information (Tables S2-S3 and Figure S1-b and -c).
L213, C214, W230, C244, L245, I246, C338, K340, F382, G383,
The specific activities of WT MAO-N and the best mutants
W430, W463 and F466], and those surrounding the extensive toward the model substrates are shown in Table 2, together with
substrate
the results of deconvoluting variants of LG-F-B7. Improved variants
were found in all cases. For example, the triple mutant LG-F-B7
(W230I/T354S/W430R) accepts the bulky substrate 2 with a specific
N
NH
H
Table 1. Grouping of the 23 chosen MAO-N residues into six
randomization sites A, B, C, D, E and F and the NDT codes used in
saturation mutagenesis. The green numbers denote “active site”
positions, the red ones “tunnel” positions and purple ones “shared”
positions.
NH
3
2
1
Scheme 1. Model amino compounds 1, 2 and 3.
Randomization site
Code
A: T93, W94, L213, C214
B: F119, F128, F382, G383
C: H142, E145, L245, I246
D: M242, C244, C338, K340
E: Y365, I367, W463, F466
F: W230, T354, W430
NDT
NDT
NDT
NDT
NDT
NDT
Figure 1. MAO-N residues chosen for saturation mutagenesis
marked in the homology model, which was built using the crystal
structure of MAO-N-D3 (PDB: 2VVL). A): Active site mutation sites
(yellow), selected on the basis of induced fit docking of amine 1
2 | J. Name., 2012, 00, 1-3
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Table 2. Specific activity (U/mg) of WT MAO-N and evolved variants in addition to mutants LG-F-B6 and LG-F-B5 (marked in bold), which
were created by deconvoluting LG-F-B7.
Entry
Mutations
1
2
3
WT MAO-N
LG-F-G6
LG-I-D11
LG-F-B7
LG-J-B4
0
0
0.13±0.01
W230R/W430C
0.108±0.001 0.23±0.02 0.04±0.002
W230R/W430C/C214L
W230I/T354S/W430R
W230I/T354S/W430R/M242R/Y365V
W230I /W430R
0.66±0.01
0.52±0.05 0.23±0.02
0.30±0.01 0.02±0.003
0.02±0.001
0.018±0.001 0.67±0.03 0.03±0.003
LG-F-B6
LG-F-B5
0.014±0.001 0.22±0.02 0.016±0.004
T354S
0.025±0.001
0
0.09±0.01
The kinetic parameters of the best MAO-N variants (LG-J-B4 and
LG-I-D11), mutant LG-F-B7 and deconvolutant LG-F-B6 toward
substrates 1 and 2 were obtained by measuring the initial velocities
of the enzymatic reaction and curve-fitting according to the
Michaelis-Menten equation. In the cases of LG-I-D11 (for amine 1)
and LG-J-B4 (for amine 2) the Vmax proved to be 1.12U/mg and
0.82U/mg (Fig S11), respectively. The data for the catalytic rate
(kcat) and catalytic efficiency (kcat/Km) are summarized in Table 3. In
the case of substrate 1, the previously evolved mutant
Asn336Ser/Ile246Met was reported to have moderate activity
(kcat=6.00min-1; kcat/Km=19.35min-1mM-1).9 In the present work,
variant LG-I-D11 (W230R/W430C/C214L) shows a 10- and 2.8-fold
improvement in kcat and kcat/Km, respectively. Additionally, the
catalytic efficiency for substrate 2 increases from LG-F-B6 and LG-F-
B7 to LG-J-B4. To the best of our knowledge, the kinetic parameters
of previous MAO-N variants in the reaction of substrate 2 have not
been reported to date.
Table 3. Kinetic characterization of the best MAO-N variants,
mutant LG-F-B7 and deconvolutant LG-F-B6.
Enzyme
Substrate Km (mM)
kcat (min−1
)
kcat/Km
(min−1
mM−1
55.15
)
LG-I-D11
LG-J-B4
LG-F-B7
LG-F-B6
1
2
2
2
1.12±0.09 61.77±1.41
1.25±0.06 46.02±0.60
1.42±0.07 37.40±0.72
1.96±0.12 21.35±0.43
36.81
26.34
10.89
We then applied the Turner-deracemization technique8-12,18 to
substrates 1 and 2 using recombinant cells of MAO-N mutants LG-I-
D11 and LG-J-B4, respectively. This procedure employs a cyclic
sequence of enantioselective oxidation with MAO-N mutants (LG-I-
D11 and LG-J-B4) and non-selective chemical reduction using
NH3·BH3 as shown in Figure 2.The deracemized products were
recovered in 73% and 86% isolated yields of (S)-1 and (S)-2,
respectively, each with high enantiomeric excess (>99% and 93.4%)
(Table 4, Figures S12, S13, S14 and S15). This is a significant result,
because thus far 1,2,3,4-tetrahydro-1-methylisoquinoline (1) could
be obtained only as the (R)-enantiomer, as shown by catalysis using
the known variant MAO-N D5.9,20 Therefore, reversal of
enantioselectivity has been achieved by introducing the present
point mutations. In order to test the generality of the mutant LG-I-
activity of 0.3U/mg, which was further improved more than 2-fold
by the introduction of two additional mutations (M242R/Y365V).
Interestingly, in the case of substrate 1, variant LG-I-D11
(W230R/W430C/C214L) shows a 6-fold increase in specific activity
relative to LG-F-G6 (W230R/W430C), and also accepts the sterically
demanding substrate
2. The most striking result of the
deconvolution experiments concerns the reaction of substrate 2.
Single mutant LG-F-B5 (T354S) shows no activity, but in concert
with the double mutant LG-F-B5 (W230I/W430R) having a specific
activity of 0.22 U/mg, the respective triple mutant
W230I/T354S/W430R displays a notable improvement (0.3 U/mg).
Thus, a strong cooperative effect is operating.19
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NH3BH3
Table 4. Scaled-up deracemization of tetrahydroisoquinolines using
best mutants LG-I-D11 and LG-J-B4 leading to (S)-products.
R
N
Enzyme
Substrate
ee (%)
Isolated yield
(%)
Imine (1, 2, 4 and 5)
R
MAO-N mutants
LG-I-D11
>99% (S)
73%
NH
NH
R
1
1 (R=methyl)
2 (R=phenyl)
4 (R=ethyl)
NH
LG-I-D11
LG-J-B4
80% (S)
81%
NH
(S)-(1,2,4 and 5)
5 (R=isopropyl)
2
93.4% (S)
86%
Figure 2. Deracemization of racemates 1, 2, 4 and 5 by employing a
cyclic sequence of enantioselective oxidation with MAO-N mutants
(LG-I-D11 and LG-J-B4) and non-selective reduction with NH3·BH3.
NH
2
D11, deracemization was also carried out for 1,2,3,4-tetrahydro-1-
ethylisoquinoline (4) and 1,2,3,4-tetrahydro-1-isopropylisoquinoline
(5). The deracemized products were recovered in 80% and 81%
isolated yields of (S)-4 and (S)-5, respectively, also with high
enantiomeric excess (>99%) (Table 4, Figures S16, S17, S18 and
S19). Compounds rac-4 and rac-5 have not been previously
subjected to MAO-N-catalyzed deracemization, in contrast to rac-2
(providing (R)-2 during deracemization).9,20 Tetrahydroisoquinolines
(THIQs) belong to a family of biologically active molecules, including
natural alkaloids and important pharmaceutical products.21
LG-I-D11
LG-I-D11
>99% (S)
>99% (S)
80%
81%
NH
4
NH
5
In order to further explore the substrate specificity, several
structurally diverse amines were subjected to MAO-N catalysis for
deracemization with LG-I-D11 and LG-J-B4 in analytical scale
reactions, respectively (Scheme 2, Figures S20-31 and Table S5)
without carrying out additional mutagenesis experiments. In some
but not in all cases excellent results were obtained. The
deracemization of amines 7, 9 and 11 with L-I-D11 resulted in 94%
ee, 99% ee and 99% ee, respectively, demonstrating high activity
and enantioselectivity. In these cases reversal of enantioselectivity
relative to the Turner results was not observed.8-10, 12b, 22 Relative to
LG-I-D11, a higher ee-value (48%) was obtained for amine 6 by
deracemization with mutant LG-J-B4. Interestingly, LG-J-B4 showed
reversed stereoselectivity for amine 7 compared with LG-I-D11. This
indicates that it should be possible to invert stereoselectivity in
general, but such a task would require further mutational studies.
In order to gain insight into the source of enhanced catalyst
activity, the ISM evolutionary process of MAO-N was analyzed using
compound 2 as substrate. We addressed this problem by applying
the Accelrys Studio 4.1 software for identifying cavities and
assessing their volume and molecular dynamics (MD) simulations
for characterizing the altered nature of the tunnel. In the first step
going from WT MAO-N to variant LG-F-B7, which was discovered in
library F, three point mutations (W230I/T354S/W430R) were
introduced, W230I (active site), T354S (tunnel), and W430R (active
site). The two deconvolutants LG-F-B6 (W230I /W430R) and LG-F-B5
(T354S) (Table 2) allowed us to identify the respective mutational
effects. The catalytic profile of deconvolutant LG-F-B6 indicates that
mutations W230I/W430R have changed the size and thus shape of
the active site pocket so that substrate 2 is accepted with notable
activity, but not in the case of the single site mutation LG-F-B5
(T354S) which occurs at the beginning of the entrance tunnel. When
comparing the shape of the computed active site pocket of WT
NH2
NH2
NH2
Cl
rac-6
rac-7
rac-8
LG-I-D11: 35% ee (R)
94% ee (R)
11% ee (S)
51% ee (R)
26% ee (R)
LG-J-B4:
48% ee (R)
NH
N
N
H
NH
rac-10
rac-11
rac-12
rac-9
LG-I-D11: >99% ee (R)
64% ee (R)
7% ee (R)
>99% ee (S)
>99% ee (S)
56% ee (S)
LG-J-B4:
0% ee
0% ee
Scheme 2. Deracemization of a panel of amine substrates using best
mutants LG-I-D11 and LG-J-B4.
MAO-N with that of LG-F-B6 (Fig. 3), it becomes clear that in the
latter variant the exchange of Trp (W) at positions 230 and 430 by
sterically smaller amino acids Ile and Arg cause severe geometric
changes. The concomitant increase in volume of the active site
pocket allows the binding of large bulky steric substrates such as 2
with two aryl moieties (volume=167 Å3).
In order to shed light on the further enhanced catalyst activity
upon going from LG-F-B6 to LG-J-B4, MD simulations were
performed in the absence of 2. This allowed the visualization of
representative conformations of variants LG-F-B4 and LG-J-B6
without any bias arising from interactions with the substrate. A
representative conformation was selected after clustering of the
phase space sampled from the 50ns MD trajectory. The
conformational change of tunnels of LG-F-B4 and LG-J-B6 is
described in Figure 4. The side-chain of M242R forms a hydrogen
bond with the oxygen atom of residue D146 which reduces the
polarity of the tunnel. The mutation Y365V results in a similar effect
at the tunnel entrance and exit (Fig. 4A). In contrast, residues D146
4 | J. Name., 2012, 00, 1-3
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Figure 4. Comparison of the tunnel surface potential of LG-F-B4 (A)
and LG-J-B6 (B). Different colors denote different levels of polarity,
deeper red coloring denoting increasing polarity.
Figure 3. Comparison of the active site volumes of WT MAO-N and
variant LG-F-B6, computed by application of Accelrys Discovery
Studio software 4.1 in the search for enzyme cavities.
and Y365 do not undergo similar interactions and consequently do
not contribute to a reduction of the polarity in the tunnel (see Fig.
4B). Overall, a decrease in polarity makes it easier for hydrophobic
substrates and products to enter and exit the enzyme, thereby
increasing the catalytic activity of LG-J-B4.23 The respective
engineered tunnels of LG-F-B4 and LG-J-B6 are shown in Figure S32
(Supporting Information). It should be pointed out that in the
present case the tunnel volume has not increased significantly upon
mutagenesis, which means that the polarity change upon going
from LG-J-B6 to LG-F-B4 constitutes the determining factor.
respectively. It is notable that the computed binding energy of LG-I-
D11-(R)-1 is approximately 6.97 kcal/mol smaller than the LG-I-D11-
(S)-1 complex. We are aware of the necessity to compute transition
state energies by quantum mechanical methods, but this was
outside the realm of the present study. At this stage we point to the
study of Sherman and coworkers who examined the relationship
between binding affinity and catalytic activity.26
With the aim of exploring the conformational changes at the
catalytic active site, the entire systems were equilibrated without
any constraints by MD for 50ns at 300K. The MD trajectory was
aligned and clustered into three clusters for each system based on
the backbone atoms of variant LG-I-D11 to identify the most
representative conformations of the sampled phase space. The
representative structures of catalytic domain from three highly
clustered conformations of MD simulation trajectory are shown in
Figure 5. The frequency of conformations in each cluster was
calculated in order to explore the possibility of emerging active
conformations. The results of the simulations show that in the LG-I-
D11-(R)-1 complex, the interatomic distance between the N-atom
of FAD and the H-atom of the stereogenic carbon in (R)-1 is
constant at a distance of ~2.1 Å (Figure 5A), a feature which
supports catalysis. We also found a similar phenomenon in the LG-I-
D11-(S)-1 complex, in this case the distance being ~2.2 Å (Fig. 5D),
but the respective frequencies are dramatically different: LG-I-D11-
(R)-1 complex (36.1%) versus LG-I-D11-(S)-1 complex (0.3%). Other
clusters are characterized by long distances as shown in Figure 5B
(4.9 Å), 5C (4.4 Å), 5E (3.6 Å) and 5F (3.3 Å). Based on the analysis of
the distance between FAD and substrate in multiple monoamine
oxidases which reveal distances of less than 2.5 Å, we conclude that
these are inactive conformations. Moreover, during the MD
simulation of the (R)-1-complex, the original conformation is
maintained (Fig. S34). This computational result is in line with the
experimentally observed stereoselectivity (Table 4).
In order to throw light on possible reasons for reversed
stereoselectivity of LG-I-D11 in the case of substrate 1, two
structural models were built for variant LG-I-D11 harboring (R)-1
and (S)-1, respectively, using the Autodock4.2 program.24 Based on
25
the previously proposed and generally accepted mechanism,
docking conformations were selected. The binding models of the
respective complexes (R)-1 and (S)-1 to LG-I-D11 are actually quite
similar (Fig S33). The benzene ring of substrate 1 is deeply
embedded in the hydrophobic area of the binding site, and the
amino-moiety points toward the FAD. This computational result is
consistent with the crystal structure of the MAO-N-D5-proline
complex (2VVM). It suggests that our LG-I-D11-(R)-1 and LG-I-D11-
(S)-1 models derived from docking are reliable and can be used for
further MD simulations. Since transformations of both (R)- and (S)-1
are catalyzed by variant LG-I-D11, a distance constraint (2.5 Å, 50
kcal/mol) between the coordinating N-atom of FAD and the H-atom
of the stereogenic C-atom in the two enantiomers was applied in
the first 5 ns of MD, thereby simulating the induced-fit process of
substrate binding. This corresponds to a pose close to the transition
state. The molecular mechanics generalized Born surface area (MM-
GBSA) method implemented in AMBER16 was used to calculate
binding free energies of the LG-I-D11-complexes of (R)- and (S)-1
after 5ns MD simulations. Table S6 shows that the MM-GBSA
predicted binding free energy (GBTOT) for the (R)- and (S)-
complexes amount to -23.69 kcal/mol and -16.70 kcal/mol,
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It is also instructive to compare the catalytic profiles of the
three variants LG-J-B4 (3 tunnel and 2 active site mutations), LG-F-
B7 (1 tunnel and 2 active site mutations) and LG-F-B6 (no tunnel
mutations, only 2 active site mutations). In the reaction of the bulky
and hydrophobic substrate 2, it can be seen that catalytic rate (kcat
)
and catalytic efficiency (kcat/Km) decrease sharply in this order
(Table 3). The evolved change in tunnel polarity supports substrate
entry and product departure. We conclude that in contrast to the
successful simultaneous mutagenesis strategy described herein,
engineering the entrance tunnel polarity and the shape of the
binding pocket sequentially involves some uncertainty and may not
be as successful. We expect the combined ISM mutagenesis
Figure 5. Representative conformations of three clusters in MD
simulations. (A) Catalytically active conformations of LG-I-D11-(R)-1;
(B) and (C) show conformations which severely restrict catalytic approach to be applicable to other enzymes in which not only the
shape of the binding pocket plays a role, but also the characteristics
of the entrance tunnel which include such properties as polarity,
shape and size.
activity; (D) Catalytically active conformations of LG-I-D11-(S)-1; (E)
and (F) show conformations which also severely restrict catalytic
activity.
Acknowledgements
Conclusions
Support from the Max-Planck-Society, the LOEWE Research Cluster
SynBioChem and Youth Innovation Promotion Association of the
Chinese Academy of Sciences (Grant No. 2016166) is gratefully
acknowledged.
We have successfully applied saturation mutagenesis for the
first time simultaneously at residues surrounding the tunnel and
lining the binding pocket of an enzyme in order to enhance activity
and to manipulate stereoselectivity. For optimal results, iterative
saturation mutagenesis (ISM) was invoked. This combined strategy
is illustrated using the monoamine oxidase from Aspergillus niger
(MAO-N) as the enzyme and structurally different amines as
Notes and references
1
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substrates.
methylisoquinoline (1) on
When
deracemizing
1,2,3,4-tetrahydro-1-
6
a
preparative scale, reversal of
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The mutagenesis strategy described herein deserves comment.
Variant LG-I-D11 is characterized exclusively by “active site”
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An efficient directed evolution strategy for enhancing activity and manipulating stereoselectivity of a monoamine oxidase is
presented.
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