Expansion of the Substrate Specificity of Porcine Kidney D-Amino Acid Oxidase for S-Stereoselective Oxidation of 4-Cl-Benzhydrylamine

Article abstract of DOI:10.1002/cctc.201800614

Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X-ray crystallographic analyses of porcine kidney D-amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of (S)-4-Cl-benzhydrylamine (CBHA) from pkDAO and obtained it by functional high-throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D-amino acids, further gaining new activity toward (S)-chiral amines with the bulky substituents. The variant enzyme (I230A/R283G) was characterized to have a catalytic efficiency of 1.85 s^?1 for (S)-CBHA, while that for (R)-1-phenylethylamine was diminished 10-fold as compared with the Y228L/R283G variant. The variant was efficiently used for the synthesis of (R)-CBHA in 96 % ee from racemic CBHA by the deracemization reaction in the presence of reducing agent such as NaBH₄ in water. Furthermore, X-ray crystallographic analysis of the new variant complexed with (S)-CBHA, together with modelling study clearly showed the basis of understanding the structure-activity relationship of pkDAO.

Full text of DOI:10.1002/cctc.201800614

Accepted Article
Title: Expansion of the substrate specificity of porcine kidney D-
amino acid oxidase for S-stereoselective oxidation of 4-Cl-
benzhydrylamine
Authors: Kazuyuki Yasukawa, Fumihiro Motojima, Atsushi Ono, and
Yasuhisa Asano
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10.1002/cctc.201800614
ChemCatChem
FULL PAPER
Expansion of the substrate specificity of porcine kidney D-amino
acid oxidase for S-stereoselective oxidation of 4-Cl-
benzhydrylamine
Kazuyuki Yasukawa[a],[b], Fumihiro Motojima[a],[b], Atsushi Ono[a], and Yasuhisa Asano *[a],[b]
Dedication ((optional))
Abstract: Discovery and development of enzymes for the synthesis
of chiral amines have been hot topics for basic and applied aspects of
biocatalysts. Based on our X-ray crystallographic analyses of porcine
kidney D-amino acid oxidase (pkDAO) and its variants, we rationally
designed a new variant that catalyzed the oxidation of (S)-4-Cl-
benzhydrylamine (CBHA) from pkDAO and obtained it by functional
high-throughput screening with colorimetric assay. The variant
I230A/R283G was constructed from the variant R283G which had
completely lost the activity for D-amino acids, further gaining a new
activity toward (S)-chiral amines with the bulky substituents. The
variant enzyme (I230A/R283G) was characterized to have a catalytic
efficiency of 1.85 s-1 for (S)-CBHA, while that for (R)-1-
phenylethylamine was diminished 10-fold as compared with the
Y228L/R283G variant. The variant was efficiently used for the
synthesis of (R)-CBHA in 96% e.e. from racemic CBHA by the
deracemization reaction in the presence of reducing agent such as
NaBH4 in water. Furthermore, X-ray crystallographic analysis of the
new variant complexed with (S)-CBHA, together with modelling study
clearly showed the basis of understanding the structure-activity
relationship of pkDAO.
racemization of the by-product (S)-1-phenylethylamine (1a) in the
presence of palladium, thereby improving the final yield of (R)-1a
to the theoretical 100%.[2] Koszelewski et al. reported the
synthesis of optically active mexiletine by the deracemization
procedure using stereoselective transaminases, although
although R- and S-selective enzymes have to be
stereocomplementary.[3]
Recently, new biocatalysts for chiral amine synthesis were
developed by directed evolution.[4a,b] Bommarius et al. have
presented the production of (R)-amines from corresponding
ketones using the developed amine dehydrogenase from already
known amino acid dehydrogenases with an NADH cofactor
regeneration system, but a short life-time of the enzyme was
observed when highly hydrophobic ketones were used as the
substrates in an aqueous-organic solvent system.[5a,b] Turner et al.
reported a deracemization reaction that uses an engineered S-
stereoselective monoamine oxidase from Aspergillus niger in the
presence of
a
reductant.[6a,b,c] The oxidase-catalyzed
deracemization method has merit in that it does not need to be
conjugated with other enzymes compared to the systems that use
amine transaminase or amine dehydrogenase.
R-stereoselective amine oxidases (AOD) that are applicable
to the deracemization of amines are a relatively recent discovery.
Engineered 6-hydroxy-D-nicotine oxidase (E350L/E352D)
catalyzing (R)-nicotine oxidation with moderate activity was
reported.[7] We have reported that a variant porcine kidney D-
amino acid oxidase (pkDAO) (Y228L/R283G) catalyzed the
synthesis of (S)-1a by deracemization. The variant completely lost
activity for D-amino acids, although the starting pkDAO has a
rather wide substrate specificity for D-amino acids.[8a,b] We solved
the structure of the variant pkDAO (Y228L/R283G) in complex
with FAD and (R)-1a to a resolution of 1.88 Å by X-ray
crystallography (PDB: 3WGT). We designed the enzyme to lose
activity for D-amino acids by removing the residues that recognize
the carboxylic acid of the substrate; made a rational explanation
for the new high activities for (R)-1a; and explained how D-amino
acids such as D-phenylalanine (3), D-methionine, and D-proline
were no longer the substrate. This variant best catalyzed the
oxidation of (R)-1a as a substrate, while a bulky substrate such
as benzhydrylamine (2a) was not compatible.
Introduction
Extensive efforts have been made on the discovery and
development of efficient biocatalysts used for the chiral amine
synthesis of building blocks for pharmaceuticals. Lipases and
transaminases have been well studied and used for the practical
kinetic resolution of amines to produce chiral amines; however,
the yields of these methods are limited because the reactions are
kinetic resolution or equilibrium reactions, respectively.[1a,b] To
overcome the limitation inherent with the use of lipase B from
Candida antarctica, Reetz and Schimossek in 1996 first
introduced the dynamic kinetic resolution reaction with a
[a]
Dr Kazuyuki Yasukawa, Dr Fumihiro Motojima, Mr. Atsushi Ono,
Prof. Dr.Yasuhisa Asano
Biotechnology Research Center and Department of Biotechnology,
Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-
0398, Japan
The aim of this study is to obtain variants of pkDAO that act
on benzhydrylamine compounds and are applicable to the
deracemization reaction to produce (R)-4-chlorobenzhydrylamine
(2c) and 4-substituted benzhydrylamine compound, which are
useful chiral building blocks for pharmaceuticals. Our strategy is
based on our experimental data from the structure-activity
relationship among the functional amino acid residues in the
E-mail: asano@pu-toyama.ac.jp
[b]
Dr Kazuyuki Yasukawa, Dr Fumihiro Motojima, Prof. Dr.Yasuhisa
Asano
Asano Active Enzyme Molucule Project, ERATO, JST, 5180
Kurokawa, Imizu, Toyama 939-0398, Japan
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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variants obtained by the saturation mutagenesis at Leu51 and
Ile215 did not show any enzyme activity toward these
substrates. A unique variant, I230V/R283G, was obtained that
also catalyzed the oxidation of 2a, but not 2c. Our result showed
that Ile230 was a very important residue to accommodate
substrates such as benzhydrylamine. On the other hand, Ile215
in pkDAO, which corresponds to Met213 in DAO from
Rhodotorula gracilis as proposed by Pollegioni et al.,[10] plays an
important role of recognizing the side chain of the substrate D-
amino acid. They actually reported that the variant M213G
efficiently catalyzed the oxidation of unnatural bulky (R)-amino
acids such as naphthyl derivatives.[10b,c] Therefore the mutation
of Ile215 in the variant I230A is expected to broaden the
substrate specificity to accommodate the bulky group of Cβ such
as α-(2-naphthyl)benzylamine.
active site of the parent and variant Y228L/R283G together with
our systematic and effective screening system.
Results and Discussion
Screening for benzhydrylamine oxidase
Recently, we described a variant (Y228L/F242I/R283G)
catalyzing the oxidation of 1-(2-naphthyl)ethylamine (kcat/Km =
0.83 s-1 mM-1) and showed a possibility of broadening the
substrate specificity based on the computational analysis of its
crystal structure.[9] Based on the crystal structure of variant
Y228L/R283G, we speculated that 4-Cl-phenyl ring of (S)-2c
would be directed toward the hydrophobic cavity formed by
Leu51, Ile215, and Ile230, and that the cavity at the active site
would play a key role in accommodating the bulky substrate.[10] It
would be possible to discriminate between the 4-Cl-phenyl group
and the non-substituted phenyl group in 2c if we carefully
choose the residues of the cavity. Therefore, the residues
Leu51, Ile215, and Ile230 in the active site of pkDAO were
chosen as targets for mutation to improve the catalytic
properties. Variants R283G and Y228L/R283G were chosen as
parents for the saturation mutagenesis of Leu51, Ile215, and
Ile230. Then, high-throughput screening by a colorimetric assay
using 96 well microtiter plates was used to determine the
oxidation activity toward 2a and (RS)-2c by the formation of red
pigment. Four variants of the mutant, I230A/R283G,
Kinetic parameter and substrate specificity
All the engineered variant enzymes were purified as
described in the experimental section. A variant with three point
mutations, Y228L/I230C/R283G, seems to lose its enzymatic
activity. This is probably because of the loss of the FAD cofactor
from the enzyme because the yellow color of the enzyme
disappeared during the purification process. The kinetic
parameters of the variant enzymes, I230A/R283G,
I230C/R283G, and I230F/R283G, were determined to evaluate
the effects of the mutations (Table 1). Compared with the variant
R283G, variants I230A/R283G, I230C/R283G, and
I230F/R283G showed lower kcat/Km values toward (R)-1a. In
contrast, the newly evolved variants were able to catalyze the
oxidation of (S)-2c. The variant I230A/R283G showed the
highest kcat/Km value toward (S)-2c amongst the variants (Table
I230C/R283G, I230F/R283G, and Y228L/I230C/R283G, were
obtained as positive clones from the screening library of
saturation mutagenesis at Ile230. All of these variants catalyzed
S-stereoselective oxidizing activity toward 2c, while all of the
Table 1. Kinetic parameter of wild-type and variants pkDAO
Wild-type
Y228L
R283G
Y228L/R283G
I230A/R283G
I230C/R283G
I230F/R283G
Km (mM)
kcat (s-1)
n.d.
n.d.
n.d.
n.d.
6.98±0.67
1.46±0.08
0.211
n.d.
7.95±0.78
9.93±0.88
1.26
7.28±0.90
0.349±0.013
0.0482
6.90±1.11
0.111±0.014
0.0161
2.40±0.30
3.42±0.30
1.45
6.25±0.89
0.0474±0.0059
0.00777
2.63±0.43
0.366±0.036
0.141
NH2
kcat/Km
(R)-1a
n.d.
n.d.
(s-1 mM-1)
Km (mM)
kcat (s-1)
n.d.
n.d.
n.d.
5.23±0.65
3.30±0.49
0.629
NH2
n.d.
n.d.
n.d.
n.d.
kcat/Km
2a
n.d.
n.d.
n.d.
n.d.
(s-1 mM-1)
NH2
Km (mM)
kcat (s-1)
n.d.
n.d.
n.d.
n.d.
2.94±0.23
6.01±0.10
2.05
2.96±0.23
5.48±0.46
1.85
3.73±0.34
4.33±0.98
1.15
n.d.
n.d.
n.d.
n.d.
Cl
kcat/Km
n.d.
n.d.
n.d.
n.d.
(s-1 mM-1)
(RS)-2c
Km (mM)
kcat (s-1)
5.01±0.27
5.19±0.09
1.04
8.41±2.24
1.72±0.01
0.215
n.d.
n.d.
n.d.
n.d.
n.d.
NH2
n.d.
n.d.
n.d.
n.d.
n.d.
COOH
(R)-3
kcat/Km
n.d.
n.d.
n.d.
n.d.
n.d.
(s-1 mM-1)
n.d.: not detected
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2). Therefore, variant I230A/R283G was selected for further
investigation. The stereoselectivity shown by variant
formed hydrophobic cavity. To explain this question, we
constructed a three-dimensional model of I230F/R283G using
MOE based on the crystal structure of variant I230A/R283G
(Figure S3). Interestingly, a shift outward in the active site of the
benzene ring of Phe230 toward Ile138 and the C1 atom of
Ile138 was observed. As a result, the enough space to accept
the chloride moiety of (S)-2c was created (Figure S3).
As a result of the studies on the substrate specificity,
I230A/R283G showed activities toward (R)-1a and (R)-1b, while
(R)-1c, (S)-1a (S)-1b, and (S)-1c were not substrates and had
negligible activities (Table 2). These results suggest that the
interaction of phenyl ring of 2c with Tyr224 as well as the
isoalloxazine ring of FAD by π-π stacking and also Phe242 by
the T-shaped stacking, which play a more important roles in
substrate recognition than the hydrophobic cavity formed by
Leu51, Ile215 and Ile230.
I230A/R283G for 2c was confirmed by using (S)- and (R)-2c.
The enzyme acted on (R)-2c with only negligible activity. The Km
values of variant I230A/R283G for (S)- and (R)-2c were 2.80
mM and 1.99 mM, respectively and their kcat values were
calculated to be 7.30 s-1 and 0.006 s-1, respectively (Table S1).
From these results, the enantiomeric ratio (E-value) for the S-
enantiomer of 2c was calculated by the following equation (1).
E-value = [kcat/Km](S-enantiomer)/[kcat/Km](R-enantiomer)
(1)
The E-value for (S)-2c was calculated to be higher than 1000,
which is enough to be utilized in the kinetic resolution of (RS)-
2c. To understand the manner of chiral discrimination toward 2c,
the variant I230A/R283G was subjected to X-ray crystallographic
analysis.
The crystal structure of variant I230A/R283G complexed
with (S)-2c was determined at 3.20 Å resolution (PDB: 5WWV).
In five out of eight molecules, the electron density corresponding
to (S)-2c was observed, even though the similar Km values for
(S)- and (R)-2c indicate that the affinities of I230A/R283G are
similar. This mutation would have made additional space to
accommodate the 4-Cl-phenyl ring of 2c, and the 4-Cl-phenyl
ring of 2c weakly interacted with Tyr228. The position of the
amine group and α-carbon of (S)-2c are similar to those of (R)-
1a in Y228L/R283G suggesting that the oxidation mechanism (a
hydride transfer) is not changed (Figure 1). [11a,b]
Docking simulation of 1b with the active site of variant
I230A/R283G supported this suggestion. (R)-1b preferred to
bind in the active site in a position similar to that of (R)-1a in the
crystal structure of Y228L/R283G, but the corresponding S-
enantiomer could not bind to anywhere in the enzyme.
Deracemization reaction using variant pkDAO (I230A/
R283G)
Optically active products have been obtained at the
theoretical yield of 100% in the oxidase-catalyzed
deracemization reaction of amines with the use of chemical
reductants such as NaBH4. Furthermore, this process has merit
since a complicated separation of the remaining substrate and
Table 2. Substrate specificity of variant pkDAO Y228L/R283G and
I230A/R283G
The reaction mixture containing the substrate, 100 mM KPB (pH 8.0), 2 mM
phenol, 1.5 mM 4-aminoantipyrine, 2 U horseradish peroxidase, 10% DMSO
and the amount of enzyme. The activity of variant Y228L/R283G toward (R)-
1a was taken as 100% (18.3 U/mg). Substrates were used at a final
concentration of 10 mM without marked as follows: [a]used at a final
concentration of 5 mM, [b] used at a final concentration of 1 mM.
Substrate
Y228L/R283G
I230A/R283G
R1
R2
(R)-1a
(S)-1a
C6H5
CH3
100 (%)
<0.1
33.6
<0.1
5.0
2.4 (%)
<0.1
0.9
Figure 1. The ligand binding in the active site of pkDAO variants.
(a) The cartoon model of pkDAO (Y228L/R283G)-(R)-1a complex. (R)-1a is
shown as ball-and-stick model colored cyan. FAD is colored in yellow. The
residues mutated for the screening of the variants oxidizing (S)-2c are
labeled and shown as stick model. (b) The superposition of pkDAO
(Y228L/R283G)-(R)-1a complex (blue) and pkDAO (I230A/R283G)-(S)-2c
complex (green). (S)-2c is shown as ball-and-stick model colored magenta.
The A-weighted Fo-Fc omit map (>4.0) of (S)-2c is shown in blue mesh.
The mutated residues are labeled. The distance of -carbon of (R)-1a and
(S)-2c to N5 atom of isoalloxazine ring are shown as orange dotted lines
(3.15 and 3.48 Å, respectively).
C6H5
CH3
(R)-1b
4-F-C6H4
4-F-C6H4
4-Cl-C6H4
4-Cl-C6H4
C6H5
CH3
(S)-1b
CH3
<0.1
<0.1
<0.1
10.9
46.5
45.2
<0.1
84.8
8.1
(R)-1c
CH3
(S)-1c
2a[a]
CH3
<0.1
n.d.
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CO2H
(RS)-2b[a]
(RS)-2c[a]
(R)-2c[a]
(S)-2c[a]
(RS)-2d[b]
(R)-3
4-F-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Br-C6H4
C6H5CH2
n.d.
n.d.
n.d.
n.d.
However, we could not explain why the variant
n.d.
I230F/R283G also showed oxidation activity toward (S)-2c,
because it was speculated that substituted phenylalanine would
hinder access of the 4-Cl-phenyl ring of (S)-2c to the newly
n.d.
n.d.
n.d.: not detected.
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the product are generally not necessary because the structure of
the starting substrate and the product is the same. The
three dimensional structure of the variant I230A/R283G
complexed with the substrate (S)-2c is a proof of concept of our
mutagenesis strategy. DAO has been widely found in eukaryotic
and prokaryotic organisms, and these enzymes showed variation
in enzymatic properties such as optimum reaction condition,
substrate specificity, and amino acid sequence. In the future, we
should be able to design several new amine oxidases evolved
from amino acid oxidases having desirable stereoselectivity for
several amines to establish the next generation of green
chemistry.
optimization of the deracemization reaction such as the effects
of buffers, pH, and reductants show that the best condition is as
follows: the reaction mixture (total volume 1 ml) contained 100
mM glycine-NaOH (pH 9.0), 5 mM (RS)-2c, 100 mM NaBH4, and
0.4 U of the purified enzyme in an aqueous medium shaken at
1.7×103 rpm at 30°C. In the optimized deracemization condition,
(R)-2c was obtained in 98.0% e.e. within 1 hr because (S)-2c
was converted to the corresponding imine-2c by pkDAO
I230A/R283G, and then the imine was immediately reduced to
(RS)-2c by NaBH4 (Figure 2). In this reaction, undesirable by-
products such as ketones and alcohols were not detected.
Previously, Turner et al. reported the deracemization of 1 mM of
2c with an S-selective monoamine oxidase variant D11C from
Aspergillus niger for 48 hr at 37°C using NH3-BH3 as a
reductant.[6b] Compared with that study, the deracemization
reaction by pkDAO I230A/R283G with the reductant NaBH4 gave
a pure (R)-2c in a short time. However, addition of too much
NaBH4 caused denaturation of the enzyme. Recently, a more
eco-friendly oxidase-catalyzed deracemization method by imine
reductase and an artificial transfer hydrogenase as a reductant
in substitution for the chemical reductant was reported.[12a,b]
However, these non-chemical reductants have limits in the
selection of the substrate; for example, only cyclic amines, which
generate stable imine under the aqueous buffer by AOD, can be
used as substrates. More recently, Aleku et al. reported the
synthesis of chiral secondary amine by unique deracemization
method using reductive amination in combination with NADP+-
regeneration system in the presence of NH3-BH3.[13] On the other
hand, an unstable imine formed from a straight chain primary
amine, such as 1a catalyzed by AOD, is quickly converted to the
corresponding ketone by aqueous hydrolysis. A control of an
unstable straight chain imine as an intermediate formed during
the deracemization reaction would be a new challenge for the
next generation of deracemization reactions.
Figure 2. Time course of the deracemization of 2c using variant
I230A/R283G
The reaction mixture (1 mL) consisted of 100 mM glycine-NaOH buffer (pH
9.0), 5 mM (RS)-2c, 100 mM NaBH4, and 0.1 U purified enzyme, and the
reaction mixture was stirred at 1.3×103 rpm at 30°C
Symbol; ●: (R)-2c, ○: (S)-2c
A preparative scale reaction was carried out for
identification of the product by the deracemization reaction using
variant I230A/R283G. The reaction product was easily purified
by extraction with hexane (40 mL × 3) under alkaline conditions,
and then, the hexane layer was evaporated in vacuo. The
resulting oil residue was further dried in a desiccator. The final
product (19.8 mg) was identified as (R)-2c (46.2% yield and
95.7% e.e.) from HPLC analysis, 1H-NMR spectra and by
examination of the optical rotation with ATAGO AP-300
polarimeter (Figure S4 and S5). Result of 1H-NMR spectra in
isolated product confirmed remaining H2O and DMSO. HPLC
analysis showed a small amount of the by-product alcohol in
deracemization reaction eluted at 3.7 min.
Experimental Section
Materials
(R)-α-1-phenylethylamine,
(S)-α-1-phenylethylamine,
(R)-4-F-α-1-
(R)-4-Cl-α-1-
(RS)-4-Cl-
phenylethylamine,
phenylethylamine,
(S)-4-F-α-1-phenylethylamine,
(S)-4-Cl-α-1-phenylethylamine,
benzhydrylamine, and benzhydrylamine were obtained from Sigma-
Aldrich (Munich, Germany). (R)-4-Cl-benzhydrylamine was purchased
from Astatech, Inc (Bristol, PA., USA). (S)-4-Cl-benzhydrylamine was
purchased from Combi-Blocks (San Diego, CA., USA). (RS)-4-F-
benzhydrylamine and (RS)-4-Br-benzhydrylamine were synthesized in our
laboratory (supporting information). All other chemicals were commercially
available.
Site-directed mutagenesis
Conclusions
Saturation mutagenesis of plasmid pDAOR283G or pDAOY228L/R283G
as a template was carried out with QuikChange Lightning Site-Directed
Mutagenesis Kit (Stratagene, California, USA), and then it was used to
transform the E. coli JM109. These mutants were cultivated at 37°C for
24 hr on an LB agar plate containing 80 µg/mL ampicillin, and then single
colonies were cultivated at 37°C for 24 hr in 100 μL LB media containing
80 µg/mL ampicillin and 1 mM IPTG in a Nunc™ DeepWell™ plate. After
We obtained a novel variant I230A/R283G from pkDAO by protein
engineering based on our X-ray crystallographic analyses of the
variant Y228L/R283G and utilized it in the synthesis of (R)-2c by
the deracemization method with NaBH4. Determination of the
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the cultivation, cells were collected by centrifugation and suspended in 100
μL 100 mM potassium-phosphate buffer (KPB) (pH 8.0). Intact cells were
suspended (10 μL) in the reaction mixture (100 μL), which consisted of 100
mM KPB (pH 8.0), 2 mM phenol, 1.5 mM 4-aminoantipyrine, 2 U
horseradish peroxidase, and a substrate, such as (R)-1a, 2a, or (RS)-2c.
The progress of the reaction was monitored by HPLC with an OD-H
column (ϕ 0.46 cm × 25 cm, Daicel Chiral Technologies Co. Ltd, Tokyo,
JAPAN) using a solvent system of hexane: 2-propanol = 9:1 (Absorbance:
220 nm, Flow rate: 1 mL/min, column temperature: 30°C). The reaction
was quenched by addition of 1M NaOH. The aqueous phase was extracted
by hexane (40 mL × 3), and then, the hexane layer was dried in vacuo.
The remaining product was analyzed by 1H-NMR and by optical rotation
measurement. The 1H NMR spectra were recorded on the Bruker Biospin
AVANCE II 400 (Bruker Biospin, Rheinstetten, Germany) system. Optical
rotation was determined by ATAGO AP-300 (Atago Co., Tokyo, Japan) by
using a 10.1-mm cell. The optical purity of the isolated (R)-2c was 95.7%
DNA sequence analysis
Nucleotide sequencing was performed using the dideoxynucleotide chain
termination method. Sequencing reactions were carried out with a BigDye®
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City,
CA., USA), and the reaction mixture was applied to an ABI PRIZM 310
Genetic analyzer (Applied Biosystems, Foster City, CA., USA).
1
e.e. with an isolation yield of 46.2% (19.8 mg, 0.091 mmol). The H-NMR
spectra were shown as follows: 2c (400 MHz; DMSO-d6) δH (ppm) 7.18-
7.43 (m, 9H), 5.09 (s, 1H), 2.33 (s, 2H). [ɑ]D27 -9.99 ° (c 1.0, EtOH) (lit. [ɑ]D
20 -10.6 (c 1.1, EtOH) for the (R)-enantiomer, [ɑ]D 20 +10.8 (c 2.18, EtOH)
for the (S)-enantiomer.). [14]
Purification of wild-type pkDAO and variant pkDAOs
All enzymes were purified by the same procedure. E. coli cells were
suspended in 5 volumes of 10 mM KPB (pH 8.0) containing 0.1% 2-
mercaptoethanol and disrupted by sonication for 20 min at 180 W using
Insonator 201M (Kubota Co., Tokyo, Japan). After the cell debris was
removed by centrifugation at 10,000 × g at 4°C for 30 min, the supernatant
was obtained as cell-free extract. The enzyme in the cell-free extract was
then fractionated by ammonium sulfate precipitation. The precipitant of 20-
33% saturation fraction was suspended in 10 mM KPB (pH 8.0) containing
0.1% 2-mercaptoethanol and dialyzed against the same buffer. The
dialyzed enzyme solution was applied to a DEAE-Toyopearl column (ϕ6.0
cm × 13 cm), and the absorbed enzyme was eluted by a linear gradient of
0-0.5 M NaCl. The enzyme solution was next saturated to 20% ammonium
sulfate and applied to a Butyl-Toyopearl column (ϕ3.0 cm × 22.0 cm). A
step-wise elution was done with 10 mM KPB buffer containing 20, 10, and
0% of ammonium sulfate saturation. The final preparation gave a single
band on SDS-PAGE. Protein concentration was measured using the
Bradford method. The specific activity for (R)-1a (20 mM) of the purified
variants R283G, Y228L/R283G, I230A/R283G, I230C/R283G, and
I230F/R283G were calculated to be 2.2, 18.3, 0.58, 0.16, and 0.071 U/mg,
respectively. The specific activity toward (R)-phenylalanine (3) of the
purified wild-type and variant Y228L were shown to be 7.5 and 0.18 U/mg,
respectively.
Acknowledgements
This work was supported by a grant from the Exploratory
Research for Advanced Technology (ERATO) Asano Active
Enzyme Molecule Project from the Japan Science and
Technology Agency (JST) (Grant Number JPMJER1102). This
research was also supported in part by a grant-in-aid for Scientific
Research S from The Japan Society for Promotion of Sciences
(No. 17H06169) to Y. Asano. We thank Dr. Kimiyasu Isobe for his
critical reading of the manuscript.
Keywords: Chiral amine • Amine oxidase • D-amino acid
oxidase • Deracemization • Oxidation
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The oxidase activity was assayed at 30°C by measuring quinoneimine dye
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phenol, 1.5 mM 4-aminoantipyrine, 2 U horseradish peroxidase, 10%
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Kinetic parameter
The kinetic parameter was determined by the same method as described
in the section of the enzyme assay using 1-20 mM (R)-1a, 1-5 mM 2a, and
1-7 mM (RS)-2c, (R)-2c, and (S)-2c. The reaction mixture contained the
substrate, 100 mM KPB (pH 8.0), 2 mM phenol, 1.5 mM 4-aminoantipyrine,
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The initial velocity was measured spectrophotometrically with the software
Spectra Manager Version
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Identification of the product by enzymatic deracemization reaction
The reaction mixture (40 mL) consisted of 100 mM glycine-NaOH buffer
(pH 9.0), (RS)-2c (50.0 mg, 0.197 mmol), 100 mM NaBH4,and 4 U purified
enzyme was stirred at 1.3×103 rpm at 30°C for 1 hr in a 1 L eggplant flask.
For internal use, please do not delete. Submitted_Manuscript
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10.1002/cctc.201800614
ChemCatChem
FULL PAPER
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Layout 1:
FULL PAPER
Newly obtained a novel D-amino acid
oxidase variant by protein engineering
and utilized it in the synthesis of (R)-4-
Cl-benzhydrylamine by the
Kazuyuki Yasukawa[a],[b], Fumihiro
Motojima[a],[b], Atsushi Ono[a], and
Yasuhisa Asano *[a],[b]
Page No. – Page No.
deracemization method with NaBH4
Expansion of the substrate specificity
of porcine kidney D-amino acid
oxidase for S-stereoselective
oxidation of 4-Cl-benzhydrylamine
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.