Asymmetric synthesis of optically active methyl-2-benzamido-methyl-3-hydroxy-butyrate by robust short-chain alcohol dehydrogenases from Burkholderia gladioli

Article abstract of DOI:10.1039/c5cc04652a

Three short-chain alcohol dehydrogenases from Burkholderia gladioli were discovered for their great potential in the dynamic kinetic asymmetric transformation of methyl 2-benzamido-methyl-3-oxobutanoate, and their screening against varied organic solvents and substrates. This is the first report of recombinant enzymes capable of achieving this reaction with the highest enantio- and diastereo-selectivity.

Full text of DOI:10.1039/c5cc04652a

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Asymmetric synthesis of optically active methyl-2-
benzamido-methyl-3-hydroxy-butyrate by robust
short-chain alcohol dehydrogenases from
Burkholderia gladioli†
Cite this: Chem. Commun., 2015,
51, 12328
Received 5th June 2015,
Accepted 22nd June 2015
Xiang Chen,ab Zhi-Qiang Liu,ab Jian-Feng Huang,ab Chao-Ping Linab and
DOI: 10.1039/c5cc04652a
Yu-Guo Zheng*ab
www.rsc.org/chemcomm
Three short-chain alcohol dehydrogenases from Burkholderia moderate enantio- and distereo-selectivity or low substrate loading
gladioli were discovered for their great potential in the dynamic (4 mM).7 Based on the discovery of SDR-producing strain Burkholderia
kinetic asymmetric transformation of methyl 2-benzamido-methyl- gladioli ZJB12126,8 we envisaged that a highly enantio- and distereo-
3-oxobutanoate, and their screening against varied organic solvents selective asymmetric synthesis of (2S,3R)-MBHB might be realized by
and substrates. This is the first report of recombinant enzymes using SDR from ZJB12126 via DYKAT (Scheme 1).
capable of achieving this reaction with the highest enantio- and
diastereo-selectivity.
To seek the appropriate enzyme for the preparation of (2S,3R)-
MBHB, three bgadh genes containing distinct sequence motifs
similar to the most representative SDR from Lactobacillus brevis
Chiral alcohols are frequently required as important and versatile (GeneBank: CAD66648.1) were identified based on the genome of
intermediates for pharmacological compounds and other fine B. gladioli BSR3 (Genbank accession no. NR_102847.1),9 cloned
chemicals. Dynamic kinetic asymmetric transformation (DYKAT) and overexpressed in Escherichia coli. These recombinant enzymes
is a powerful tool for accessing single enantiomers with a theore- (BgADH1, BgADH2, and BgADH5) displayed 31–34% sequence
tical yield of 100%.1 Many short-chain alcohol dehydrogenases/ identities with each other, and owned common ‘‘Rossmann-fold’’
reductases (SDRs) have been exploited and subjected to the DYKAT motifs, cofactor-binding domains covered N-terminal TGXXXGXG,
of racemic ketones.2 However, upscale DYKAT was hampered due NNAG and PG, and N–S–Y–K catalytic tetrads (ESI,† Fig. S3).10 With
to the limited commercially available enzymes, narrow substrate the codon optimization of the bgadh2 gene sequence, the expres-
scope, and poorly soluble substrates.3 Thus, there is substantial sion level of BgADH2 was significantly improved (ESI,† Fig. S2).
interest in developing robust enzymes and extending the applica- After purification, the protein concentration of optimized BgADH2
tion of DYKAT in stereo-selective synthesis.
(2.6 mg mLÀ1) was enhanced approximately 3-fold compared to
The presence of (2S,3R)-methyl-2-benzamido-methyl-3-hydroxy- its counterpart (0.9 mg mLÀ1). The molecular subunit mass of
butyrate (MBHB) in (3R,4R)-4-acetoxy-3-[(R)-(t-butyl dimethylsilyloxy)- BgADHs was around 27 kDa and the quaternary structures of
ethyl]-2-azetidinone (4-AA), an important building block for carba- BgADH1, BgADH2, and BgADH5 were tetramers.
penems, is well-documented.4 Achieving this complex compound
with excellent enantio- and diastereo-selectivity is normally a difficult
task.5 The DYKAT of methyl-2-benzamido-methyl-3-oxobutanoate
(BMOB) using a transition metal catalyst has been established, but
it requires high-pressure and leaves trace metal contamination in
the product.6 In an attempt to develop a renewable alternative to this
target reaction, an enzymatic method with ambient pressures and
no production of toxic waste is urgently required. Several enzymes
have been developed to give (2S,3R)-MBHB, but with low to
a Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014,
China. E-mail: zhengyg@zjut.edu.cn; Fax: +86-571-88320630;
Tel: +86-571-88320630
b Engineering Research Center of Bioconversion and Biopurification of Ministry of
Education, Zhejiang University of Technology, Hangzhou 310014, China
Scheme 1 Asymmetric synthesis of enantioenriched MBHB by SDR via
† Electronic supplementary information (ESI) available: Tables S1–S7, Fig. S1–S38,
dynamic kinetic asymmetric transformation.
and the detailed experimental procedure. See DOI: 10.1039/c5cc04652a
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Table 1 DYKAT of BMOB using different recombinant enzymesa
Enzyme
Cf
ee (%)
de (%)
Cv (%)
Activityb (U mgÀ1
)
BgADH1
BgADH2
BgADH5
CpSCR
LbADHc
RDRc
(2S,3R)
(2S,3R)
(2S,3R)
(2S,3R)
(2S,3R)
(2S,3R)
(2S,3R)
(2S,3R)
1
99
99
99
99
99
94
60
99
99
69
99
92
89
89
49
99
99
51
99
—
—
—
—
3.65
5.09
2.62
4.37
—
—
—
FPDHc
RAXc
—
a
Reaction conditions: BMOB (40 mM, DMSO, 5% v/v), NADP+ (0.4 mM),
glucose (5%, w/v), purified enzyme (0.1 mg mLÀ1), GDH (0.1 mg mLÀ1),
pH 6.5, and 30 1C. Activities were measured under the standard assay
protocol. Cf, configuration; Cv, conversion. Data from ref. 7c.
b
Fig. 1 Effect of organic solvents on the biosynthesis of (2S,3R)-MBHB
using purified BgADH2. The amounts of organic solvents added were 25%
(v/v) for water-miscible solvents and 50% (v/v) for water-immiscible solvents.
Activities were measured under the standard assay protocol. The activity in
the absence of organic solvent was taken as 100%. DMSO, dimethyl sulfoxide;
DMF, dimethylformamide; IPA, iso-propanol; THF, tetrahydrofuran; EtOAc,
ethyl acetate; BuOAc, butyl acetate; iBuOAc, iso-butyl acetate; CH2Cl2,
dichloromethane; c-hexane, cyclohexane.
c
BgADH2 was found to display excellent stereo-selectivity,
affording the corresponding (2S,3R)-MBHB with 99% ee and
98.5% de (Table 1). BgADH1 and BgADH5 catalysed this trans-
formation as well yet with low enantio-selectivity or moderate
diastereo-selectivity. In comparison, BgADH2 showed unique and
the highest enantio- and distereo-selectivity yielding (2S,3R)-MBHB
reported so far. Notably, the KmNADPH values of purified BgADHs
were 3- to 22-fold lower than that against NADH, emphasizing
that BgADHs preferred NADPH over NADH. The overall catalytic
efficiency (kcat/KmBMOB) of BgADH2 (12.3 sÀ1 mMÀ1) was found
to be the highest as compared to BgADH1 (5.83 sÀ1 mMÀ1),
BgADH5 (2.81 sÀ1 mMÀ1), and CpSCR (10.0 sÀ1 mMÀ1) from
Candida parapsilosis (GeneBank: GQ411433.1) (ESI,† Table S6).11
Although the well-known LbADH exhibited strong Mg2+ dependency,9a
BgADHs were typical of non-metal SDRs (ESI,† Table S5). BgADH2
activity was enhanced 10-fold in response to different temperatures,
and the optimal temperature and pH were at 30 1C and 6.5. A
half-life of more than 48 h was found at 30 1C for BgADH2,
suggesting that it was rather robust and suitable for prolonged
incubation. The employment of BgADH2 also exhibited excellent
functions under the ‘‘enzyme-coupled’’ cofactor regeneration
using glucose dehydrogenase (GDH) and glucose,12 avoiding
the reversibility and poor thermodynamic driving force in the
‘‘substrate-coupled’’ approach.13
The effect of organic solvents, that played a crucial role in
biocatalysis toward poorly soluble susbtrates,14 on the activity and
stereo-selectivity of BgADH2 using BMOB as the substrate was
analyzed in-depth. In the presence of water-miscible solvents, an
insignificant negative effect was observed on the activity of BgADH2,
except for acetone, likely because of the subunit dissociation, cofactor
desorption, or the tendency of hydrophilic solvents to strip tightly
bound water in the enzyme active site.15 Similarly, the stereo-
selectivity of BgADH2 was impaired in hydrophilic solvents, except
for DMSO and DMF.16 This might be relieved by immobilization,
which can improve enzymatic performance in organic solvents.17
When the water-immiscible solvents were added, the relative activities
were roughly correlated with the log Po/w values of solvents, except for
dichloromethane,18 and remained completely intact in high log Po/w
solvents, such as n-hexane and iso-octane (Fig. 1).16 Importantly,
BgADH2 retained excellent enantio- and diastereo-selectivity as well as
high relative activity in the presence of toluene with higher partition
efficiencies for BMOB and (2S,3R)-MBHB.
A further study of the developed aqueous-toluene biphasic
system for (2S,3R)-MBHB production to increase substrate
solubility and avoid product inhibition demonstrated better
productivity than that in the monophasic system (Fig. 2a). With
a highly optimized process development, full conversion was
achieved with a 60 mM substrate in short times by the driving
force of the acidity of the buffer that faciliated the racemization
of (2R)-BMOB through the achiral enol.19 A plateau emerged
only after 2 h and the final production of (2S,3R)-MBHB was
55.1 mM (yield 91.1%) in 99% ee and 98.5% de (Fig. 2b).
To reveal the substrate recognition and catalytic principle of
BgADHs, a set of structurally diverse aldehydes and ketones were
subjected to the reduction conditions (Table 2). It was noteworthy
that BgADHs followed the anti-Prelog’s rule with R preference and
worked well toward varied aryl ketones and ketoesters, except for 20,
21, 22, and 33. This might be explained by the small hydrophobic
active site tunnels in BgADHs, and thus the hydrophilic groups or
macromolecules were impeded embedding in the hydrophobic
cavity.20 The inverted stereochemical assignment toward 25–28, 32,
and 34 was due to the smaller side of the ketone having a higher
Cahn–Ingold–Prelog priority than the large one. The Prelog stereo-
preference of BgADH2 against 14 and 18 might be the presence of the
hydrogen bond formed between substituents and enzyme, modifying
the enzymatic properties.21 Electron-withdrawing substituents
Fig. 2 (a) Effects of substrate concentrations on the (2S,3R)-MBHB production
with BgADH2 in monophasic and biphasic systems. (b) Time course of
(2S,3R)-MBHB production using BgADH2. Three independent measure-
ments were taken for each point.
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Table 2 Substrate specificities of BgADHs from B. gladioli ZJB12126
Fig. 3 Binding models of BgADH2 with BMOB. (a) Detailed view of the
interactions between BgADH2 and (2S)-BMOB; (b) detailed view of the
interactions between BgADH2 and (2R)-BMOB.
The molecular basis of BgADH2 in the binding interaction of
BMOB 29 was predicted, wherein the CQO oxygen of (2S)-BMOB
forms hydrogen bonds with both Y151 and S138 (2.0 and 1.9 Å)
and it is protonated from Y151-OH, followed by the attack of a
hydride from C4-NADPH toward the CQO carbon of (2S)-BMOB
(3.6 Å) (Fig. 3a), in accordance with the proposed catalytic
mechanism of SDR.3b,10,20a This hypothesis was further supported
by the site-directed mutagenesis. Mutation of Y151 or S138 to A
resulted in an almost complete (499%) loss of enzyme activity.
Overall, NADPH provides its C4-hydride to attack the si-face of
(2S)-BMOB, giving rise to the (R)-product, which is consistent with
the experimentally observed (2S,3R)-MBHB.22
In summary, we have successfully developed a renewable
alternative to the asymmetric reduction of BMOB via enzymatic
DYKAT, affording an enantio- and distereomerically pure (2S,3R)-
MBHB. BgADH2 from B. gladioli ZJB12126 was the first enzyme with
excellent enantio- and distereo-selectivity for (2S,3R)-MBHB produc-
tion (99% ee, 98.5% de). An aqueous-toluene biphasic system
significantly enhanced the substrate loading (60 mM) and produc-
tivity (91.1%). BgADH2 also exhibited a broad substrate scope and
followed the anti-Prelog’s rule, especially toward aryl ketones and
ketoesters. Docking provided insight into the understanding of the
molecular basis of BgADH2, useful for further engineering of this
enzyme for other enantio-selective transformations.
BgADH1
BgADH2
BgADH5
Activity
Cf eea
(%)
Activity
Cf eea
(%)
Activity
Cf eea
(U mgÀ1
) (%)
S
(U mgÀ1
)
(U mgÀ1
)
1
2
3
4
5
6
7
8
9
0.32
0.50
7.90
1.15
1.32
1.52
0.74
1.62
2.50
—
—
—
—
—
—
—
—
1.33
1.05
8.45
1.15
1.37
2.59
0.85
1.62
3.03
6.35
6.21
6.57
4.83
5.19
6.56
6.98
6.13
6.76
3.52
3.35
3.64
2.02
5.56
4.47
5.68
5.62
4.12
3.82
5.09
—
—
—
—
—
—
—
—
0.32
0.51
5.74
0.82
1.05
1.29
0.85
1.14
1.96
4.51
4.39
4.63
3.64
4.03
4.66
5.35
4.05
4.98
2.32
—
—
—
—
—
—
—
—
—
—
—
—
10 6.13
11 5.49
12 6.29
13 4.18
14 5.11
15 6.28
16 6.44
17 5.67
18 6.22
19 2.43
R(99)
R(99)
R(99)
R(93)
R(99)
R(99)
R(99)
R(98)
R(87)
—
—
—
—
R(499)
R(499)
S(95)
S(99)
S(98)
S(85)
2S,3R(0)
R(99)
R(499)
R(99)
R(95)
S(99)
R(99)
R(99)
R(99)
S(499)
—
R(41)
R(98)
R(90)
R(499)
R(499)
S(499)
S(99)
S(499)
S(99)
R(90)
R(499)
R(95)
R(90)
R(99)
R(99)
R(99)
R(99)
R(90)
—
20
21
22
—
—
—
—
—
—
—
—
23 4.47
24 3.92
25 5.14
26 4.71
27 3.02
28 3.91
29 3.65
30 2.55
31 3.02
32 1.40
4.15
4.06
4.59
4.27
2.42
3.37
R(499)
R(499)
S(86)
S(77)
S(99)
S(15)
2S,3R(99)
3R,5R(98)
3R,5S(97)
S(37)
—
This study was financially supported by the National Basic
Research Program of China (973 Program) (No. 2011CB 710800) and
Natural Science Foundation of Zhejiang Province (No. R3110155).
2S,3R(99) 2.63
3R,5R(95) 2.55
3R,5S(96) 3.12
3R,5R(86) 3.11
3R,5S(88) 3.33
S(499)
—
—
1.36
—
1.09
S(499)
—
S(99)
1.13
—
—
33
34
—
—
—
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