Accessing d-Valine Synthesis by Improved Variants of Bacterial Cyclohexylamine Oxidase

Article abstract of DOI:10.1002/cctc.201701229

Chemoenzymatic deracemization was applied to prepare d-valine from racemic valine ethyl ester or l-valine ethyl ester in high yield (up to 95 %) with excellent optical purity (>99 % ee) by employing a newly evolved cyclohexylamine oxidase (CHAO) variant Y321I/M226T exhibiting catalytic efficiency that was 30 times higher than that of the wildtype CHAO. Interestingly, CHAO and its variants showed opposite enantioselectivity for valine ethyl ester and phenylalanine ethyl ester.

Full text of DOI:10.1002/cctc.201701229

Accepted Article
Title: Novel Access to D-Valine Synthesis by Improved Variants of
Bacterial Cyclohexylamine Oxidase
Authors: Rui Gong, Peiyuan Yao, Xi Chen, Jinhui Feng, Qiaqing Wu,
Peter C. K. Lau, and Dunming Zhu
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10.1002/cctc.201701229
ChemCatChem
COMMUNICATION
Novel Access to D-Valine Synthesis by Improved Variants of
Bacterial Cyclohexylamine Oxidase
Rui Gong, [a] [b] Peiyuan Yao, *[a] Xi Chen, [a] Jinhui Feng, [a] Qiaqing Wu, *[a] Peter C. K. Lau, *[a] and
Dunming Zhu [a]
Abstract: Chemo-enzymatic deracemization was applied to prepare
D-valine from racemic valine ethyl ester or L-valine ethyl ester in high
isolated yield (up to 95%) and excellent optical purity (ee > 99%)
employing a newly evolved cyclohexylamine oxidase (CHAO) variant
Y321I/M226T that exhibited 30 times higher catalytic efficiency than
the wild type CHAO. Interestingly, CHAO and its variants showed
opposite enantioselectivity for valine ethyl ester and phenylalanine
ethyl ester.
Turner et al. reported a deracemization strategy for the
[13]
preparation of primary, [11] secondary, [12] and tertiary amines
[14]
as well as substituted pyrrolidines
employing monoamine
oxidase from Aspergillus niger (MAO-N) mutants. Similarly,
based on the substrate profile and crystal structure analysis of
cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans
strain IH-35A by Lau et al., [15] we have extended the substrate
scope of CHAO to more bulky amines by protein engineering
and examined the biocatalytic potential of these CHAO mutants.
[16]
Although this chemo-enzymatic deracemization strategy has
D-Amino acids are important building blocks in pharmaceuticals,
agrochemicals and food additives. [1] D-Valine in particular, exists
widely in a variety of agricultural pesticides, semisynthetic
veterinary antibiotics and pharmaceutical drugs, such as
been demonstrated as an effective approach for the preparation
of optically pure amines, it has not been applied in
deracemisation of racemic amino acid esters for the preparation
of D-amino acids such as D-valine. Herein we explored the
feasibility of preparing D-valine from racemic valine ethyl ester or
L-valine ethyl ester employing the wild type CHAO (wt CHAO)
and its genetic variants (Scheme 1). The improved CHAO
mutants were also tested toward other amino acid derivatives to
determine their activity and enantioselectivity.
fluvalinate, valnemulin, penicillamine, actinomycin D, fungisporin
[2]
and valinomycin.
It can also selectively inhibit fibroblasts
proliferation in cell culture. [3] Therefore, investigation of efficient
synthetic methods for D-valine is of high importance. Current
state of the art approaches for the synthesis of D-valine includes
chemical resolution, chemical asymmetric synthesis, and
[2]
enzymatic transformation, each has its shortcomings.
Chemical resolution is commonly used in industry,
[4]
but it
requires expensive chiral resolving agents and complex
experimental process. Chiral auxiliaries, such as (R)- or (S)-4-
phenyl-2-oxazolidinone, in chemical asymmetric synthesis are
[5]
expensive.
Compared to chemical methods, enzymatic
preparation of D-valine is an environmentally friendly process
with high stereoselectivity and mild reaction conditions. To date,
many enzymatic methods have been developed, for example,
asymmetric degradation of DL-valine by L-amino acid oxidase; [6]
stereoselective hydrolysis by D-stereospecific amidohydrolase; [7]
Scheme 1. Deracemization of racemate ethyl valinate by employing CHAO
mutant and NH3·BH3.
specific hydrolysis of
DL-5-isopropylhydantoin by
D-
Previously, the application of CHAO in biocatalysis has been
focused on the deracemisation of primary and secondary
amines to produce chiral amine building blocks in high yield and
enantiomeric excess (ee). [16] In this work, we initially found that
L-valine ethyl ester was a better substrate compared to L-valine,
but the activity of the wt CHAO was not high (0.9 U/mg).
hydrantoinase coupled with N-carbamoyl-D-amino acid
[8]
amidohydrolase;
and asymmetric synthesis from 2-oxo-3-
[9]
methylbutyric acid by D-amino acid aminotransferase
amino acid dehydrogenase. [10]
or D-
We thus sought for CHAO variants that may have an
improved activity by modelling L-valine ethyl ester into the active
site of CHAO. [15a] Effectively, 11 amino acid residues (F88, T198,
L199, M226, Q233, Y321, F351, L353, F368, P422, Y459) lining
the binding pocket of CHAO were selected and mutated to six
typical amino acids (Ala, Ile, Phe, Trp, Thr, Tyr) by site-specific
mutagenesis (Figure S1 in the Supporting Information). The
resultant mutants were assayed against L-valine ethyl ester. [17]
As a result, four substitutions (T198I, L199F, M226T and Y321I)
were found to possess a significantly improved catalytic activity
compared to the wt CHAO enzyme (Table S1 in the Supporting
Information). In order to obtain better mutants toward L-valine
ethyl ester, iterative mutagenesis was performed on the best
mutant Y321I. Consequently, three double mutants, Y321I/T198I,
Y321I/L199F, and Y321I/M226T were created and assayed. The
best mutant Y321I/M226T was selected as template for the last
[a]
R. Gong, Prof. P. Yao, Prof. X. Chen, Prof. J. Feng, Prof. Q. Wu,
Prof. P.C.K. Lau, Prof. D. Zhu
National Engineering Laboratory for Industrial Enzymes
Tianjin Engineering Research Center of Biocatalytic Technology
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences
32 Xi Qi Dao, Tianjin Airport Economic Area
Tianjin 300308 (P.R. China)
Fax: (+86) 22-24828703
E-mail: yao_py@tib.cas.cn
wu_qq@ tib.cas.cn
peter.lau@tib.cas.cn
R. Gong
University of Chinese Academy of Sciences
No.19(A) Yuquan Road, Shijingshan District, Beijing, 100049
(P.R.China)
[b]
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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10.1002/cctc.201701229
ChemCatChem
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round of mutagenesis resulting in
Y321I/M226T/L199F, and Y321I/M226T/T198I/L199F.
Y321I/M226T/T198I,
study (Figure 1) showed that the reaction was fast and linear in
the first two hours, resulting in up to 99% and 77% ee for mutant
Y321I/M226T. The deracemization process of L-valine ethyl
ester plateaued in the next one hour, with a modest increase in
ee to 99%. As a result, D-valine ethyl ester was isolated after 4 h,
and hydrolyzed to D-valine by 2 mol/L of HCl solution. After
removal of the solvent, D-valine hydrochloride was obtained in
95% and 91% yield. Although the reaction was also fast and
linear in the first two hours for CHAO, resulting in 58% and 19%
ee, respectively, the ee leveled out at a lower value (62% and
64% ee).
The kinetic parameters of the wt CHAO and its variants
against substrate L-valine ethyl ester showed that three of the
single amino acid substitutions (T198I, L199F and Y321I)
displayed an improved catalytic efficiency (2-13 folds) whereas
that of M226T was marginal compared to the wt CHAO (Table
1). According to the structure of CHAO, [15a] M226 is located at
the entrance of the tunnel distal to the active site. Hence the
M226T mutation may have a low influence on the activity.
Interestingly, T198I, L199F, and Y321I all have high kcat values
but their high Km in 5 mM range resulted in a sharp decline of
catalytic efficiency. In contrast, the catalytic efficiency of the
double mutant Y321I/M226T was 30 fold higher than the wt
CHAO. Interestingly, iterative mutagenesis of M226T and Y321I
yielded triple (Y321I/M226T/T198I) and quadruple mutants
(Y321I/M226T/T198I/L199F) that possessed higher kcat than
other mutants, but their high Km values resulted in lower catalytic
efficiencies.
Table 1. Kinetic parameters of wt CHAO and mutants toward L-valine
ethyl ester.
kcat / Km
(min-1mM-1)
Enzyme[a]
wt CHAO
Km (mM)
7.5
kcat (min-1)
38.5
5.1
Figure 1. Time course of deracemization of D/L-valine ethyl ester and L-valine
ethyl ester by employing CHAO or its mutant Y321I/M226T and borane–
ammonia complex. D/L-valine ethyl ester with CHAO (■) or its mutant
Y321I/M226T(▲), L-valine ethyl ester with CHAO (●) or its mutant
Y321I/M226T(★).
T198I
5.0
65.2
13.0
10.6
6.5
L199F
5.2
55.3
M226T
5.1
33.3
To shed light on the possible molecular mechanism of the
catalysis, we performed docking experiments. Docking D/L-valine
ethyl ester to the wt CHAO generated several binding poses. In
the D-docking position, the Cα and NH of valine ethyl ester are
4.31 Å and 4.75 Å from the N (5) of FAD. The O (4) of FAD
forms a hydrogen-bonding interaction with NH of valine ethyl
ester, so the NH moved and deviated from the above of N (5) of
FAD as shown in Figure 2 (a). In the L-docking position (Figure
2 (b)), the Cα and NH of valine ethyl ester are 4.22 Å and 4.44 Å
from the N (5) of FAD. Y321 has a hydrogen-bonding interaction
with NH of valine ethyl ester, so it places NH right above the N
(5) of FAD. Therefore the L-enantiomer is favored, whereas the
activity toward D-enantiomer is below detection.
Y321I
1.1
75.0
66.4
72.2
152.2
77.5
38.2
Y321I/ T198I
Y321I/ M226T
Y321I/ M226T/T198I
Y321I/M226T/T198I/F199F
1.8
130.7
186.2
212.6
221.4
1.2
2.7
5.8
Docking of D-valine ethyl ester to the mutant Y321I/M226T
showed a similar result as the wt CHAO that gave negligible
activity (Figure 2 (c)). The best docking result was obtained by
L-valine ethyl ester (Figure 2 (d)). Here, the Cα and NH of L-
valine ethyl ester are 3.75 Å and 3.13 Å from the N (5) of FAD.
The hydrogen-bonding interaction formed between O (4), N (5)
of FAD and NH of L-valine ethyl ester with other interaction force
pull them closer and these interactions anchor the L-valine ethyl
ester and promote stability. These attributes evidently led the
[a] Y321I/L199F and Y321I/ M226T/L199F failed to be expressed
Then we applied the Turner-deracemization technique [11, 13b,
13d, 18]
to racemic valine ethyl ester and L-valine ethyl ester by
using E. coli whole cells of CHAO and its double mutant
Y321I/M226T in combination with the nonselective chemical
reducing agent (NH3·BH3) at pH 6.5 and 30 oC. The time course
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10.1002/cctc.201701229
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mutant to show higher activity and affinity to the substrate with
an overall 30-fold increase in catalytic efficiency.
was D-enantiomer. In contrast, L-enantiomer was the selectivity
of the mutants toward alanine ethyl ester and valine ammonia
amide. These results showed that the enzymes displayed
opposite enantioselectivity toward aromatic compounds.
In order to further confirm the enantioselectivity of mutant
Y321I/M226T toward phenylalanine ethyl ester, deracemization
of racemic phenylalanine ethyl ester was performed by
employing mutant Y321I/M226T and borane-ammonia complex
in 10 mL reaction. The results indicated that L-phenylalanine
was obtained in up to 99% ee value within 8 h (Figure S6 and
S7 in the Supporting Information).
To elucidate the possible basis for the dramatic reversal of
enantioselectivity, protein–ligand docking simulations using the
structure of mutant Y321I/M226T indicated that D-phenylalanine
ethyl ester adopted a binding mode where the benzene ring
pointed out of the active site, whereas the benzene ring of L-
phenylalanine ethyl ester pointed toward the active site (Figure
3). The latter state resulted in an increase of distance between
NH of phenylalanine ethyl ester and N (5) of FAD from D-
phenylalanine ethyl to L-phenylalanine ethyl. Consequently, the
benzene ring was postulated to exert an important influence on
the enantioselectivity of the enzyme. Valinamide and 2-
aminobutanamide have the similar structure but in fact the
enzyme has opposite enantioselectivity, it is an interesting result
and worth further research.
Figure 2. Docking structure of (a) D-valine ethyl ester and (b) L-valine ethyl
ester in the active site of wt CHAO; Docking structure of (c) D-valine ethyl ester
and (d) L-valine ethyl ester in the active site of mutant Y321I/M226T.
Table 2. Specific activity (U/mg)[a] of wt CHAO and mutants.
Substrate
CHAO
Y321I
Y321I/M226T
D-tyrosine ethyl ester
L-tyrosine ethyl ester
D-phenylalanine ethyl
ester
L-phenylalanine ethyl
ester
D-alanine ethyl ester
L-alanine ethyl ester
D-prolinamide
Trace[b]
0.029
0.014
0.019
0.016
ND[c]
0.188
0.018
0.705
1.043
0.013
ND[c]
0.022
0.117
ND[c]
ND[c]
0.012
0.106
ND[c]
ND[c]
ND[c]
0.141
ND[c]
ND[c]
L-prolinamide
D-valine ammonia
amide
ND[c]
Trace[b]
ND[c]
L-valine ammonia
amide
0.011
0.016
0.03
D-phenylglycinamide
L-phenylglycinamide
L-2- aminobutanamide
D-2-aminobutanamide
0.023
Trace[b]
ND[c]
0.021
Trace[b]
ND[c]
0.034
0.012
ND[c]
0.016
0.016
0.022
[a] One enzyme unit (U) was defined as the amount of enzyme that produced
1 μmol of hydrogen peroxide per minute. [b] Trace: activity below 0.01 U/mg.
[c] ND: not detected
A series of amino acid derivatives were examined as the
substrates of the wt CHAO and its mutants Y321I and
Y321I/M226T. Each substrate was assayed individually at 10
mM substrate concentration with the purified enzymes (Table 2).
In the case of D-phenylalanine ethyl ester the activity of the
double mutant Y321I/M226T improved significantly compared to
those of the wt CHAO and mutant Y321I. Interestingly, wt CHAO
and Y321I had inverse enanioselectivity toward tyrosine ethyl
ester, but Y321I/M226T showed no selectivity. It is possible that
residues lining the active site and entrance tunnel can influence
enantioselectivity. In all cases, there was no activity towards the
enantiomers of prolinamide. The enantioselectivity of both
mutants toward phenylalanine ethyl ester and phenylglycinamide
Figure 3. Docked structures of D-phenylalanine ethyl ester (dull-red) and L-
phenylalanine ethyl ester (yellow).
In summary, a new enzyme variant of the flavoprotein CHAO
was found useful for the synthesis of D-valine in high yield and
enantiomerically pure form via the deracemization and
stereoinversion of valine ethyl ester properties that are otherwise
difficult to obtain by previously reported enzymatic methods. In
addition, CHAO and its variants showed opposite
enantioselectivity for valine ethyl ester and phenylalanine ethyl
ester. Further protein engineering of CHAO is underway in our
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This work was financially supported by the Youth Innovation
Promotion Association of the Chinese Academy of Sciences
(Grant No. 2016166) and National Natural Science Foundation
of China (Grant No. 21302215). Support by Tianjin “Thousand
Talents” Program for Senior International Scientists to P.C.K.
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Keywords: Deracemization • D-Valine • Monoamine Oxidase •
Stereoinversion • D-Amino Acids
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Rui Gong, Peiyuan Yao*, Xi Chen,
Jinhui Feng, Qiaqing Wu*, Peter C. K.
Lau*, and Dunming Zhu
Page No. – Page No.
Novel Access to D-Valine Synthesis
by Improved Variants of Bacterial
Cyclohexylamine Oxidase
Chemo-enzymatic deracemization of D/L-valine ethyl ester and L-valine ethyl ester
was applied to prepare D-valine in high isolated yield and excellent optical purity
employing CHAO mutant.
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