Reshaping the Active Pocket of Amine Dehydrogenases for Asymmetric Synthesis of Bulky Aliphatic Amines

Article abstract of DOI:10.1021/acscatal.7b04135

The asymmetric reductive amination of ketones with ammonia using engineered amine dehydrogenases (AmDHs) is a particularly attractive and environmentally friendly method for the synthesis of chiral amines. However, one major challenge for these engineered AmDHs is their limited range of accepted substrates. Herein, several engineered AmDHs were developed through the evolution of naturally occurring leucine dehydrogenases, which displayed good amination activity toward aliphatic ketones but restricted catalytic scope for short-chain substrates. Computational analysis helped identify two residues, located at the distal end of the substrate-binding cavity, that generate steric hindrance and prevent the binding of bulky aliphatic ketones. By fine-tuning these two key hotspots, the resulting AmDH mutants are able to accept previously inaccessible bulky substrates. More importantly, the mutations were also proved applicable for expanding the substrate scope of other homologous AmDHs with sequence identities as low as 70%, indicating a broad effect on the development of AmDHs and the synthesis of structurally diverse chiral amines.

Full text of DOI:10.1021/acscatal.7b04135

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Article
Reshaping the Active Pocket of Amine Dehydrogenases
for Asymmetric Synthesis of Bulky Aliphatic Amines
Feifei Chen, Gao-Wei Zheng, Lei Liu, Hao Li, Qi Chen, fulong li, Chun-Xiu Li, and Jian-He Xu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04135 • Publication Date (Web): 15 Feb 2018
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Reshaping the Active Pocket of Amine Dehydrogenases for
Asymmetric Synthesis of Bulky Aliphatic Amines
FeiꢀFei Chen†, GaoꢀWei Zheng†, Lei Liu, Hao Li, Qi Chen, FuꢀLong Li, ChunꢀXiu Li, and JianꢀHe Xu*
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State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East Chiꢀ
na University of Science and Technology, Shanghai 200237, China
ABSTRACT: The asymmetric reductive amination of ketones with ammonia using engineered amine dehydrogenases (AmDHs) is
a particularly attractive and environmentally friendly method for the synthesis of chiral amines. However, one major challenge for
these engineered AmDHs is their limited range of accepted substrates. Herein, several engineered AmDHs were developed through
the evolution of naturally occurring leucine dehydrogenases, which displayed good amination activity toward aliphatic ketones but
restricted catalytic scope covering shortꢀchain substrates. Computational analysis helped identify two residues, located at the distal
end of the substrateꢀbinding cavity, that generate steric hindrance and prevent the binding of bulky aliphatic ketones. By fineꢀtuning
these two key hotspots, the resulting AmDH mutants are able to accept previously inaccessible bulky substrates. More importantly,
the mutations were also proved applicable for expanding the substrate scope of other homologous AmDHs with sequence identities
as low as 70%, indicating a broad impact on the development of AmDHs and the synthesis of structurally diverse chiral amines.
KEYWORDS: Amine dehydrogenase, reductive amination, chiral amine, protein engineering, biocatalysis, substrate scope
11
(AADHs),10 and the construction of a chimeric AmDH by
INTRODUCTION
Chiral amines are important building blocks frequently used in
domain shuffling of the two engineered enzymes. These engiꢀ
neered AmDHs enable the direct synthesis of chiral amines
the synthesis of numerous biologically active molecules in the
from ketones (without any carboxyl group) using a coupled
pharmaceutical and agrochemical industries. According to a
glucose dehydrogenase (GDH) or formate dehydrogenase
recent estimation, approximately 40% of active pharmaceutical
ingredients (APIs) approved by the US Food and Drug Adminꢀ
(FDH) cofactor recycling system (Scheme 1).
istration (FDA) in recent years contain one or more chiral
amine moieties.1 During recent decades, considerable effort
has been dedicated to the development of more efficient and
sustainable chemical methods for the synthesis of chiral
amines, as exemplified by the progress in classic asymmetric
hydrogenation of imines/enamines and the emergence of some
C–C and C–N bond formation methods. However, these chemꢀ
ical strategies suffer from many problems such as harsh reacꢀ
tion conditions, the use of precious and environmentallyꢀ
unfriendly transition metal catalysts, poor stereoselectivity,
and tedious amino protection and deprotection processes.2
Scheme 1. Amine Dehydrogenaseꢀcatalysed Asymmetric Reꢀ
ductive Amination of Ketones with Ammonia.
With advances in protein engineering technologies, bioꢀ
catalysis has emerged as a promising and environmentally
Inspired by the engineering strategy of Bommarius and colꢀ
benign alternative to traditional chemocatalysis for the syntheꢀ
leagues, ourselves and another group successfully developed a
sis of optically pure amines.3 Numerous biocatalytic routes
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respective AmDH from Exiguobacterium sibiricum
and
catalysed by various enzymes including lipases,4 ωꢀ
transaminases,5 amine oxidases,6 lysases,7 imine reductases,8
reductive aminases,9 and amine dehydrogenases (AmDHs)10–17
have been successfully developed for the synthesis of chiral
amines. Among them, AmDHꢀcatalysed asymmetric reductive
amination of prochiral ketones has attracted much interest as it
can directly produce chiral amines using inexpensive ammonia
as amino donor. Although a wildꢀtype (WT) AmDH from
Streptomyces virginiae with reductive amination activity has
been reported, the stereoselectivity was poor and thus far the
corresponding gene/protein sequence has not been disclosed.18
Recently, Bommarius and coworkers first reported the develꢀ
opment of two engineered AmDHs after several rounds evoluꢀ
tion of naturally occurring amino acid dehydrogenases
Rhodococcus sp. M4.13 By combining our engineered AmDH
with a screened alcohol dehydrogenase (ADH) with poor enanꢀ
tioselectivity, we subsequently constructed a redoxꢀneutral
twoꢀenzyme cascade for asymmetrically transforming racemic
secondary alcohols into chiral amines.12 Notably, a similar
concept, dualꢀenzyme hydrogenꢀborrowing cascade, was also
proposed and more systematically investigated by Turner and
coworkers.14 More recently, Mutti and coworkers demonstratꢀ
ed the synthetic potential of the engineered AmDHs by conꢀ
ducting a thorough investigation on their catalytic repertoire,
optimal reaction conditions and stereoselectivity using a varieꢀ
ty of ketones and aldehydes.15 The applicability of AmDH was
further substantiated by a potent biphasic reaction system emꢀ
ploying a more thermostable AmDH from Caldalkalibacillus
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thermarum with substrate loading up to 400 mM.16 In addition,
different enzyme immobilisation techniques also proved to be
applicable to AmDHs with facile operation, elevated catalytic
performance and lower catalyst cost.17
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Table 1. Kinetic Parameters and Half-lives of AmDHs En-
gineered from Naturally Occurring Leucine Dehydrogen-
ases (LeuDHs)a
1
2
3
4
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7
8
enzyme
Km (mM)
kcat (s–1)
kcat/Km (mM–1 s–1) t1/2 (min)b
However, to date, only a limited number of AmDHs have
been developed through engineering of existing WT AADHs
as scaffolds. Moreover, one major issue with the engineered
AmDHs is their narrow substrate scope. Expanding the subꢀ
strate scope of engineered AmDHs based on structural inforꢀ
mation is therefore a major aim of many researchers. In the
present study, we aimed to probe new residues that may affect
the binding of bulky aliphatic ketones with AmDHs, and subꢀ
sequently tailor them to expand the substrate scope of this
class of enzymes.
EsAmDH
LfAmDH
BspAmDH
9.2 ± 0.3c
30.6 ± 2.0
32.4 ± 1.7
0.84 ± 0.01c
1.7 ± 0.04
2.2 ± 0.04
0.091c
0.057
0.069
450
2190
540
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aFor determining kinetic parameters, activity was measured in
NH4Cl/NH4OH buffer (2 M, pH 9.5) containing 0.1 mM NADH and varied
concentration of 2ꢀpentanone at 30°C. bHalfꢀlife (t1/2) was determined by
incubating purified enzyme at 60°C and measuring the residual activity
toward 2ꢀpentanone over time. cData from reference.12
RESULTS AND DISCUSSION
LfAmDH was built based on the crystal structure of a leucine
dehydrogenase from Bacillus sphaericus (PDB: 1LEH), which
shares 86% sequence identity with LfAmDH. By docking the
substrate 2ꢀheptanone into the active site of LfAmDH, we reꢀ
vealed that the substrateꢀbinding cavity cannot accommodate
relatively bulky aliphatic substrates (Figure 1A). Based on the
binding mode shown in Figure 1A, we assumed that residue
A113 located at the distal end of the substrateꢀbinding cavity
may generate steric hindrance that prevents the binding of
bulky ketones. Consequently, we initially mutated A113 to the
smaller glycine. As anticipated, the single site mutation
(LfAmDHꢀM1, A113G) resulted in a remarkable expansion of
the substrate specificity to include longerꢀchain aliphatic keꢀ
tones (Table 2, entries 1 and 2). Compared with the starting
LfAmDH, LfAmDHꢀM1 displayed a 13ꢀfold increase in specifꢀ
ic activity toward 2ꢀhexanone, and the emergence of activity
(425 mU/mg) against the longer 2ꢀhepanone. To further conꢀ
firm our assumption, a saturation mutagenesis library comprisꢀ
ing all 20 natural amino acids at position 113 was constructꢀ
ed.20 The results of activity assays show that only the
LfAmDHꢀM1 (A113G) mutant exhibited improved activity
toward 2ꢀhexanone, while other mutants displayed significant
decreases in activity and activity was completely lost in some
cases, suggesting the replacement of A113 with bulky amino
acids is detrimental to the activity of LfAmDH (Figure S2).
Development of AmDHs by Engineering the Existing
AADHs. Using a group of naturally occurring leucine dehyꢀ
drogenases (LeuDHs) available in our laboratory as starting
templates,19 several functional AmDHs with measurable activiꢀ
ty toward aliphatic ketones were developed by introducing the
same mutations (K68S/N261L) reported by Bommarius and
colleagues (Table S2).10a Among them, three amine dehydroꢀ
genases, namely EsAmDH from Exiguobacterium sibiricum,12
LfAmDH from Lysinibacillus fusiformis, and BspAmDH from
Bacillus sphaericus, exhibited both considerable catalytic effiꢀ
ciency and impressive thermostability (Table 1). LfAmDH
possessed the longest halfꢀlife (t1/2 = 36.5 h) at 60°C, and exꢀ
hibited the highest reductive amination activity (up to 3 U/mg
protein) toward aliphatic ketones containing four or five skeleꢀ
tal carbon atoms, but an extremely low activity toward bulky
aliphatic ketones such as 2ꢀhexanone and no activity at all for
some substrates such as 2ꢀheptanone and 2ꢀoctanone (Table
S3). Considering the limited substrate scope of the engineered
AmDHs, further engineering to create new AmDH mutants
with broader specificity is highly desirable. Herein, LfAmDH
was chosen as a representative enzyme for investigation of
substrate scope.
Expanding the Substrate Scope of Engineered AmDHs.
To more rationally identify the residues affecting the binding
of bulky aliphatic ketones in AmDHs, a homology model of
Figure 1. Illustration of the substrateꢀbinding cavities of LfAmDH (A) and its mutant LfAmDHꢀM3 (A113G/T134G) (B) with
docked substrate 2ꢀheptanone. Residues surrounding the substrateꢀbinding pocket are shown in surface representation, and 2ꢀ
heptanone (2ꢀHep) is shown as ballꢀandꢀstick model. Residues 113 and 134 responsible for steric hindrance, are shown as blue
sticks in the wildꢀtype LfAmDH and red sticks in mutant LfAmDHꢀM3 (A113G/T134G), respectively. The two catalytic residues
(K80 and D115) and the main two residues initially mutated to generate AmDH activity (L261 and S68) are shown as orange and
green sticks, respectively.
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Inspired by this result, we speculated that other residues surꢀ
mM–1 s–1, and 0.247 mM–1 s–1 for LfAmDHꢀM1, LfAmDHꢀM2,
and LfAmDHꢀM3, respectively). Moreover, the enhancement
in kcat/KM is mainly attributed to an increase in kcat (Figure 2B
and 2C).
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rounding the substrateꢀbinding pocket may also generate steric
hindrance and thereby prevent binding of bulky ketones. Alaꢀ
nineꢀscanning mutagenesis of the region surrounding G113
site was performed to identify other potential ‘hotspots’ for
further expanding the substrate specificity, and T112, E114,
T134 and V294 were selected as potential ‘hotspots’ for Alaꢀ
nine mutagenesis (Figure S3). Mutagenesis of these residues
and subsequent activity assays of the resulting mutants reꢀ
vealed that A113G/T134A (LfAmDHꢀM2) displayed a 3.3ꢀfold
further increase in specific activity (1400 mU/mg) toward 2ꢀ
heptanone compared to A113G (LfAmDHꢀM1) (Table 2, en-
tries 2 and 6). Notably, residue Thr134 is also located at the
distal end of the substrateꢀbinding cavity (Figure 1A), and
thus is also likely to be a key residue for expanding the subꢀ
strateꢀaccessible space of LfAmDH. Subsequently, we further
mutated A134 to the smaller glycine, and the resultant
LfAmDHꢀM3 (A113G/T134G) mutant displayed even higher
activity than the LfAmDHꢀM2 (A113G/T134A) mutant (Table
2, entries 6 and 7).
The two crucial mutations, A113G and T134G, resulted in
an obvious enlargement in the substrateꢀbinding cavity, and
consequently, the larger 2ꢀheptanone substrate can be well
accommodated in a relatively extended conformation (Figure
1B). This observation can be further supported by measuring
the increased volume of substrateꢀbinding pocket, which reꢀ
vealed that the volume of LfAmDHꢀM3 is 43 Å3 larger than
that of the starting enzyme LfAmDH (Supporting
Information).21 These results demonstrate that spaceꢀ
generating mutations can create additional substrateꢀbinding
volume in the binding cavity of LfAmDH which is beneficial
to reduce steric hindrance and expand the range of accepted
substrates.
Given that there is sometimes a tradeꢀoff between high enꢀ
zymatic activity and enzyme stability, we determined the meltꢀ
ing points (Tm) of the engineered mutants by circular dichroꢀ
ism measurements (Figure S4). Excitingly, all three mutants
displayed high thermostability (Tm = 67.9°C, 70.0°C, and
68.1°C for LfAmDHꢀM1, LfAmDHꢀM2, and LfAmDHꢀM3,
respectively), similar to the starting LfAmDH (Tm = 68.5°C).
The Tm value of LfAmDHꢀM2 was even 1.5°C higher than that
of the starting LfAmDH. These results suggest that the acquisiꢀ
tion of the new enzymatic activity did not have a detrimental
effect on stability, which is beneficial for the practical applicaꢀ
tion of these mutants in chiral amine synthesis.
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Table 2. Specific Activity of LfAmDH and Its Mutants to-
ward Aliphatic Ketones with Varied Chain Lengths
specific activity (mU mg–1 protein)a
entry
mutant
2ꢀpentanone
930 ± 13
220 ± 3
2ꢀhexanone
110 ± 6
2ꢀheptanone
n.a.b
1
2
3
4
5
6
7
LfAmDH
A113G
1470 ± 80
920 ± 30
360 ± 8
430 ± 0.8
300 ± 3
A113G/T112A
A113G/E114A
A113G/V294A
A113G/T134A
A113G/T134G
270 ± 1.1
57 ± 3
170 ± 10
430 ± 40
1400 ± 26
2590 ± 160
130± 2
770 ± 50
1560 ± 30
2240 ± 160
340 ± 19
670 ± 38
Figure 2. Comparison of kinetic parameters for LfAmDH,
LfAmDHꢀM1 (A113G), LfAmDHꢀM2 (A113G/T134A), and
LfAmDHꢀM3 (A113G/T134G) toward 2ꢀpentanone, 2ꢀ
hexanone, and 2ꢀheptanone. (A) Comparison of kcat/KM. (B)
Comparison of kcat. (C) Comparison of KM. Kinetic constants
of LfAmDH toward 2ꢀheptanone were not measured due to the
very low activity.
aActivity was measured in NH4Cl/NH4OH buffer (2 M, pH 9.5) containing 0.1
mM NADH and 20 mM substrate at 30ºC. bn.a. = no measurable activity.
Kinetic Parameters and Thermostability. To comparaꢀ
tively evaluate catalytic efficiency, kinetic parameters of the
starting LfAmDH and its mutants were determined using 2ꢀ
pentanone, 2ꢀhexanone, and 2ꢀheptanone (Figure 2). It is
shown that all mutants (LfAmDHꢀM1, LfAmDHꢀM2, and
LfAmDHꢀM3) exhibited a great improvement in catalytic effiꢀ
ciency (kcat/KM) toward 2ꢀhexanone and 2ꢀheptanone compared
with the starting LfAmDH (Figure 2A). For 2ꢀheptanone,
LfAmDH showed no detectable activity, while its three muꢀ
tants exhibited a progressive increase in kcat/KM with the enꢀ
largement of substrateꢀaccessible space (0.038 mM–1 s–1, 0.184
Exploration of Substrate Scope. We further examined the
activities of these mutants against a panel of ketones with difꢀ
ferent carbon chain lengths, including linear alkyl ketones,
branched alkyl ketones, and a representative alkene ketone
(Figure 3). The starting LfAmDH displayed good activity toꢀ
ward aliphatic ketones 1 and 2 that have fewer than six carbon
atoms in the skeleton, but very low or even no measureable
activity toward bulky substrates. By contrast, its three mutants
with enlarged substrateꢀbinding pockets displayed much
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Figure 3. Reductive amination activity of LfAmDH and its mutants toward aliphatic ketones with different carbon chain lengths.
Table 3. Reductive Amination of Aliphatic Ketones with
Varied Carbon Chain Lengths by LfAmDH and its Mu-
tantsa
improved activity toward longerꢀchain aliphatic ketones. For
LfAmDHꢀM3 with the largest substrateꢀbinding cavity, a carꢀ
bon chain length of up to 10 atoms was accepted, and activity
toward substrates 6−10 was observed, albeit at very low levels
for 2ꢀnonanone and 2ꢀdecanone (14 mU mg–1 and 2.5 mU mg–1,
respectively).
conversion
(%)b
ee (%)
entry
substrate
enzyme
(Config.)b
1
2
LfAmDH
LfAmDHꢀM1
LfAmDHꢀM2
LfAmDHꢀM3
LfAmDH
>99
86
97
98
99
>99
>99
>99
4
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
n.d.c
Analytic Scale Synthesis of Chiral Amines. The reactivity
of LfAmDH and its three mutants was assayed using ketones
with varied carbonꢀchain lengths as substrates (Table 3). The
results show that the starting LfAmDH exhibited great reactiviꢀ
ty toward shortꢀchain aliphatic ketones such as 2ꢀpentanone
and 2ꢀhexanone but only trace conversion toward 2ꢀheptanone
(entry 9) and even no measurable conversions toward longerꢀ
chian aliphatic ketones (entries 13 and 17), while the mutants
displayed not only excellent conversion toward shortꢀchain
ketones but also considerable or emerging conversions toward
bulky aliphatic ketones. Among these mutants, LfAmDHꢀM3
afforded the highest conversions in the amination of bulky
aliphatic ketones. Furthermore, all mutants maintained the
enantioselectivity of the starting LfAmDH, and yield (R)ꢀ
configuration products with >99% ee, suggesting that the
spaceꢀgenerating mutations in the binding cavity do not affect
the specific orientation of the substrates in the pocket.22 To
synthesize the enantiomeric amines with an opposite configuꢀ
ration, (S)ꢀselective AmDHs need to be developed. We specuꢀ
late that (S)ꢀselective AmDHs might be accessible by either
inverting the stereoselectivity of (R)ꢀAmDHs or evolving the
existing Dꢀamino acid dehydrogenases through protein engiꢀ
neering techniques.23
Synthesis of (R)-2-Heptanamine on a 10 mL Scale. The
reductive amination of 2ꢀheptanone catalysed by LfAmDH and
its mutants was scaled up to 10 mL to further evaluate the feaꢀ
sibility of the biocatalytic process (Figure 4). The three pockꢀ
etꢀextended mutants continued to display pronounced activity
toward 2ꢀheptanone, while the starting LfAmDH exhibited
extremely low reactivity and afforded only <5% conversion
within 24 h, in line with the results in Table 3 (entries 9−12).
LfAmDHꢀM3 displayed the highest reaction rate toward 2ꢀ
heptanone, reaching >97% conversion within 6 h and >99%
conversion within 12 h, followed by LfAmDHꢀM2 that
achieved >93% conversion within 6 h and >99% conversion
within 12 h. LfAmDHꢀM1 also achieved almost full converꢀ
2ꢀpentanone
3
4
5
6
LfAmDHꢀM1
LfAmDHꢀM2
LfAmDHꢀM3
LfAmDH
2ꢀhexanone
2ꢀheptanone
2ꢀoctanone
2ꢀnonanone
7
8
9
10
11
12
13
14
15
16
17
18
19
20
LfAmDHꢀM1
LfAmDHꢀM2
LfAmDHꢀM3
LfAmDH
99
>99
>99
0
LfAmDHꢀM1
LfAmDHꢀM2
LfAmDHꢀM3
LfAmDH
45
61
80
0
>99 (R)
>99 (R)
>99 (R)
n.d.c
LfAmDHꢀM1
LfAmDHꢀM2
LfAmDHꢀM3
0
n.d.c
12
22
>99 (R)
>99 (R)
aReaction conditions: 1.5 mg AmDH (purified protein), 4 mg FDH (crude cellꢀ
free extract), 50 mM substrate, 1 mM NAD+, DMSO (2% v/v for 2ꢀpentanone
and 2ꢀhexanone, 4% v/v for 2ꢀheptanone, 2ꢀoctanone and 2ꢀnonanone),
NH4COOH/NH4OH buffer (1 M, pH 8.8), 1 mL total volume, 30ºC, 180 rpm
c
for 24 h. bDetermined by GC analysis (Supporting Information). n.d. = not
detected due to low activity.
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sion (99%) within 24 h, albeit at a lower initial reaction rate
sequence identity with LfAmDH, respectively) to achieve the
same results, suggesting their general applicability in expandꢀ
ing the catalytic scope of other homologous AmDH enzymes.
Table 4. Specific Activity (mU mg–1 protein) of EsAmDH
and BspAmDH Mutants toward Aliphatic Ketones with
Varied Carbon Chain Lengthsa
1
2
3
4
5
6
7
8
than LfAmDHꢀM2 and LfAmDHꢀM3, indicating that all three
mutants are robust under the reaction conditions. Moreover,
the continual transformation of ketone to amine during the
initial 12 h implies that these mutants possess considerable
operational stability under the reaction conditions, which is in
agreement with their high Tm values.
mutation
2ꢀpentanone
800 ± 30
220 ± 20
240 ± 14
460 ± 25
1030 ± 17
240 ± 20
380 ± 2
2ꢀhexanone
83 ± 0.4
2ꢀheptanone
n.a.b
EsAmDH
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57
58
59
60
EsAmDHꢀA122G
820 ± 40
1000 ± 40
1430± 60
89 ± 1.1
350 ± 20
700 ± 60
1480 ± 25
n.a.b
EsAmDHꢀA122G/T143A
EsAmDHꢀA122G/T143G
BspAmDH
BspAmDHꢀA113G
1290 ± 20
1340 ± 30
2360 ± 10
370 ± 20
1350 ± 14
2800 ± 90
BspAmDHꢀA113G/T134A
BspAmDHꢀA113G/T134G
750 ± 9
Figure 4. Time courses of reductive amination of 2ꢀheptanone
by LfAmDH and its mutants.
aActivity was measured under the same conditions as described in Table 2. bn.a.
= no measurable activity.
Validation of the Identified Hotspots in Other AmDHs.
To evaluate the general applicability of these two identified
hotspots (A113G and T134G) for expanding the substrate
scope, these mutations were engineered into the other two
aforementioned AmDHs, EsAmDH from Exiguobacterium
sibiricum and BspAmDH from Bacillus sphaericus, which
share 70.1% and 95.6% amino acid sequence identity with
LfAmDH, respectively. Satisfyingly, incorporation of these
two spaceꢀgenerating mutations also enabled the acceptance of
bulkier aliphatic ketones, and significantly enhanced activity
toward them (Table 4). In accordance with the results of the
LfAmDH mutants, the A113G/T134G (A122G/T143G in
EsAmDH) mutation in BspAmDH resulted in the greatest inꢀ
crease in activity toward longerꢀchain aliphatic ketones, while
mutation of A113G (A122G in EsAmDH) and A113G/T134A
(A122G/T143A in EsAmDH) also markedly enhanced the
enzyme activity. These results confirm that the identified
hotspots can be manipulated in other AmDHs sharing adequate
homology with LfAmDH to create new AmDH mutants with
significantly elevated activity toward bulky aliphatic ketones.
Therefore, this substrateꢀbinding space generating mutagenesis
approach might be an effective and universal approach for
expanding the catalytic scope of homologous AmDHs in a
minimalist fashion.
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
Chemicals, mutagenesis, protein expression and purification,
enzyme assay and screening, kinetic analysis, enzyme stability
studies, protein homoꢀmodeling and molecular docking, bioꢀ
transformation, analytic method, GC chromatograms, NMR
analysis.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: jianhexu@ecust.edu.cn.
Author Contributions
†The authors contributed equally to this work (F.ꢀF. Chen and G.ꢀ
W. Zheng).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was financially supported by the National Natural Sciꢀ
ence Foundation of China (Nos. 21472045 and 21536004).
CONCLUSIONS
We successfully identified two key residues surrounding the
substrateꢀbinding pocket in AmDHs that affect the binding of
bulky aliphatic ketones, and subsequently developed several
AmDH mutants with significantly increased substrate acꢀ
ceptance by tailoring these two key sites using the starting
AmDH as template. The best mutant displayed significantly
enhanced catalytic efficiency toward bulky aliphatic ketones
with more than six atoms in the carbon skeleton such as 2ꢀ
heptanone and 2ꢀoctanone, without compromising the robust
stability and excellent stereoselectivity (>99% ee) of the WT
enzyme. More significantly, these key residues can be manipuꢀ
lated in other two AmDHs (sharing 70% and 96% amino acid
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