One-Pot Enzymatic Synthesis of d-Arylalanines Using Phenylalanine Ammonia Lyase and l-Amino Acid Deaminase

Article abstract of DOI:10.1007/s12010-018-2794-3

The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic d,l-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure d-arylalanine, a modified AvPAL with high d-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for d-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual l-enantiomer product in reaction solution could be converted into the d-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH₃BH₃. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (ee_D)) of d-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize d-arylalanine with different groups on the phenyl ring. Among these d-arylalanines, the yield of m-nitro-d-phenylalanine was highest and reached 96%, and the ee_D exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

Full text of DOI:10.1007/s12010-018-2794-3

Appl Biochem Biotechnol
https://doi.org/10.1007/s12010-018-2794-3
One-Pot Enzymatic Synthesis of D-Arylalanines Using
Phenylalanine Ammonia Lyase and L-Amino
Acid Deaminase
1
1
1
Longbao Zhu & Guoqiang Feng & Fei Ge
&
1
1
2
1
Ping Song & Taotao Wang & Yi Liu & Yugui Tao
&
3
Zhemin Zhou
Received: 22 March 2018 /Accepted: 23 May 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the
amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer
arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-
arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of
catalytic mechanism and structure, the Asn347 residue in the active site was proposed to
control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A,
the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than
that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction
solution could be converted into the D-enantiomer product through stereoselective oxidation
by PmLAAD and nonselective reduction by reducing agent NH BH . At optimal conditions,
3
3
the conversion rate of t-CA and optical purity (enantiomeric excess (ee )) of D-phenylalanine
D
reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a
broad range of substrate and could be used to efficiently synthesize D-arylalanine with different
groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine
Fei Ge contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-018-
2794-3) contains supplementary material, which is available to authorized users.
*
Yi Liu
myputer@163.com
1
2
3
School of Biochemical Engineering, Anhui Polytechnic University, 8 Zheshan Road, Wuhu, Anhui
241000, People’s Republic of China
Key Laboratory of Food and Biotechnology, School of Food and Biotechnology, Xihua University,
Chengdu 610039, China
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology,
Jiangnan University, Wuxi 214122, People’s Republic of China

Appl Biochem Biotechnol
was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using
AvPAL and PmLAAD has prospects for industrial application.
.
.
.
Keywords Phenylalanine ammonia lyase L-Amino acid deaminase D-Arylalanine One-pot
.
.
synthesis Gene cloning and expression Mutation
Background
D-Arylalanines are useful intermediates in the production of pharmaceuticals, including β-
lactam antibiotics, small peptide hormones, and pesticides. The types of pharmaceuticals
synthesized from D-arylalanines include analgesics, antistress agents, antidiabetics (e.g.,
nateglinide), and anticoagulants [1, 2]. With the increasing demand for D-arylalanies, chemical
[3], fermentative [4], and enzymatic methods [5], have already been developed to synthesize
D-arylalanines. In contrast with the chemical and fermentative methods, which suffer from
process complexity, high cost, low yield, and environmental pollution, enzymatic methods are
most suitable for industrial manufacture with regard to their optical purity and productivity and
offer an efficient, highly specific and environmentally friendly alternative to chemical and
fermentation methods. Various enzymatic approaches have been developed to achieve scalable
processes for preparing D-arylalanines, including the use of D-amino acid aminotransferase
(
DAAT) [6, 7], L-amino acid oxidase (LAAO) [8, 9], and hydantoinase [10]. However, the
operation process using aminotransferase is complex due to presence of an external cofactor
PLP) regeneration system, and the product consists of α-keto acids and D-arylalanines; the
(
separation of this mixture presents a major limitation of the use of the DAAT-catalyzed
synthesis. The LAAO-catalyzed method has been used to produce optical D-arylalanines
through the asymmetric resolution of the racemic DL-arylalanines, and the method afforded a
5
0% theoretical yield. The hydantoinase method is the primary method used for the commer-
cial production of D-amino acids, and the synthetic process followed is shown in Scheme S1
Electronic Supplementary Material) [11, 12]. In this process, the DL-5-monosubstituted
(
hydantoin as feedstock is hydrolyzed using hydantoinase, and the resulting N-carbamoyl-D-
amino acid is subsequently hydrolyzed by carbamoylase to yield the D-arylalanines stereospe-
cifically. However, the carbamoylase reaction is the rate-determining step in the process and
suffers from low enzyme activity and instability in application [13]. Moreover, the feedstock,
DL-5-monosubstituted hydantoin, which is produced through the enzymatic racemization of L-
5
-monosubstituted hydantoin, is not accessibly obtained. The racemization rate of the L-5-
monosubstituted hydantoin by hydantoin racemase is very low. For industrial production,
significant consideration must be given to the availability of feedstock, the optical purity of
products, and the enzymatic catalysis steps. From an industrial point of view, the availability of
cheap feedstock and the development of enzyme catalysts suitable for feedstock are the most
important considerations. Since trans-cinnamic acid (t-CA) is commercially produced at low
costs [14], their direct use as feedstock, would lead to a simple and efficient D-arylalanine
synthesis process, which would be expected to improve yields and be economically beneficial.
Phenylalanine ammonia lyase (PAL) can catalyze the reversible addition of ammonia to t-CA
+
at high concentration of NH4 to produce phenylalanine. PAL is a member of the 4-methylene-
imidazol-5-one (MIO)-dependent enzyme family, which includes PAL, histidine ammonia-lyase
[15] (HAL), tyrosine ammonia-lyase [16] (TAL), and phenylalanine and tyrosine aminomutases

Appl Biochem Biotechnol
[17, 18] (PAM and TAM, respectively). MIO is a highly electrophilic prosthetic group that is
formed post-translationally and autocatalytically by condensation of a highly conserved Ala-Ser-
Gly motif. The catalytic process was proposed as E1cB mechanism, the MIO electrophilically
+
attacks an NH4 group of the substrate to form a covalent adduct MIO-NH , which facilitates the
2
+
addition of NH4 group at C and protonation of C by a nearby tyrosine base located at 78 sites
α
β
in the active site of the enzyme [19, 20] (Electronic Supplementary Material, Fig. S2). Since PAL
from Anabaena variabilis exhibits higher stability and broader substrate scope than other PALs
[21, 22], it can use t-CA or its derivatives as readily available starting materials [23–25], does not
rely on cofactor recycling system, and achieves a 100% theoretical yield, a synthetic process using
AvPAL has excellent prospects in industry. However, the reaction product is a racemic DL-type
mixture resulting from the low stereoselectivity of AvPAL, which exhibits nearly equal L-
selectivity and D-selectivity. A modified AvPAL with high D-setreoselectivity is expected. Al-
though two excellent mutants H359Y and H359K with high D-selectivity were obtained using
high-throughput screening method [26], we aim to find other key amino acids which manipulate
the stereoselectivity of AvPAL through structure-and mechanism-based analysis and synthesize
highly pure D-arylalanines using one-pot enzymatic synthesis process. The synthesis course was
referred to method of Parmeggiani [26], the residual l-enantiomer was converted into the D-
enantiomer through stereoselective oxidation by PmLAAD and nonselective reduction by the
reducing agent NH BH . However, the MIO as an electrophilic group was inactivated by the
3
3
nucleophile, such as sodium borohydride and NH BH , the t-CA or t-CAs with electron-donating
3
3
group (such as methyl, hydroxyl) in phenyl ring could not be converted to phenylalanine in the
presence of NH BH . Only t-CAs with electron-withdrawing groups (such as nitro, fluoro) can be
3
3
converted to corresponding D-phenylalanine derivatives in the presence of NH BH [26]. Until
3
3
now, it is not clear why the inactivated MIO by borohydride can catalyze the t-CAs with electron-
withdrawing groups in phenyl ring. Therefore, in order to expand the application in synthesis of
highly pure D-arylalanines, we modified the process of Parmeggiani [26] to catalyze t-CA and its
derivatives with electron-donating group in phenyl ring to produce D-arylalanines. In this work,
the t-CA and derivatives were first converted to racemic arylalanines using AvPAL, and then, the
PmLAAD and NH BH were added into reaction system, the L-products were sequentially
3
3
converted to D-products, which resolve the deactivation of MIO by NH BH . As a result, the
3
3
conversion ratio of t-CA reached 82% and the optical purity (ee ) of D-phenylalanine exceeded
D
99%. Furthermore, the one-pot method can be used to catalyze the synthesis of D-arylalanines
from trans-cinnamic acid with different groups in the phenyl ring, the process was effective in
producing high purity D-arylalanines.
Experimental Methods
Vectors, Strains, Genes, and Materials
Escherichia coli JM109, E. coli BL21, and expression vector pET28a were purchased from
Novagen. The phenylalanine ammonia lyase gene from Anabaena variabilis and the L-amino
acid deaminase gene from Proteus mirabilis were synthesized commercially by Sangon
Biotech (Shanghai, China). trans-Cinnamic acid and its derivatives were obtained from
Sigma-Aldrich. All restriction enzymes (BamHI, NotI, NdeI), T DNA ligase, and DNA
4
polymerase were purchased from TaKaRa (Japan). The commercial kits for plasmid extraction,
gel extraction, and DNA extraction were purchased from Sangon Biotech (Shanghai, China).

Appl Biochem Biotechnol
Plasmid Construction
The PAL gene (pal) from Anabaena variabilis (NCBI, LF643444.1) and the PmLAAD gene
laad) from Proteus mirabilis (NCBI, EU669819.1) were synthesized commercially and
(
inserted into the pUC19 vector to produce cloned pUC19-pal and pUC19-laad plasmids.
The pal construct was designed with flanking restriction sites (BamHI and NotI) for subse-
quent subcloning and expression in the pET28a vector. The pUC-pal and pET28a vectors were
digested simultaneously with BamHI and NotI. Then, the pal gene was ligated into the pET28
vector using T4 DNA ligase at 16 °C for 12 h to produce the expression plasmid pET28a-pal.
Because PmLAAD is a membrane-bound protein and to achieve soluble expression in E. coli,
the gene sequence of laad from P. mirabilis was modified by removing one N-terminal
transmembrane region (from 21st to 87th nucleotide), the plasmids pET28a-laad was con-
structed by the previous described methods [27, 28]. The two expression plasmids pET28a-pal
and pET28a-laad were transformed into E. coli JM109 and subsequently grown on Luria-
Bertani (LB) agar plates containing kanamycin (50 μg/mL). The two plasmids were then
isolated from the cells and successful subcloning was confirmed by sequencing.
Site-Directed Mutagenesis
The mutants were generated using PCR. The PCR reaction was conducted using the PrimeSTAR
HS DNA Polymerase (Takara, Japan) and the pET28a-pal plasmid as the template DNA. The
primer sequences (altered nucleotides underlined in parentheses) are 5′-AACTCAGT
C A C C G ATA A C ( G C C ) C C A C TA AT T- 3 ′ a n d 5 ′ - AT C A A C AT C A AT TA
GTGG(GGC)GTTATCGG T-3′. After PCR, the template DNAwas digested using DpnI (Takara,
Japan). The PCR products (pET28a-pal-N347A) were first transformed into E. coli JM109 cells
for sequencing and then into E. coli BL21 cells after plasmid preparation for enzyme expression.
Expression and Purification
The three recombinant plasmids pET28a-pal, pET28a-pal-N347A, and pET28a-laad were
transformed into E. coli BL21 and grown in Luria-Bertani medium with kanamycin (50 μg/
mL). The culture was incubated at 37 °C until the OD600 reached 0.4–0.6. Then, the expression
was induced by addition of 0.4 mmol/L IPTG to the cells followed by incubation at 24 °C for
2
4 h. The cells were collected by centrifugation at 4 °C and resuspended using 50 mmol/L
Tris-HCl buffer (containing 10 mmol/L imidazole and 150 mmol/L NaCl at pH 7.5) and
disrupted by sonication on ice. The extract was centrifuged at 15,000×g for 10 min, and the
supernatant was used for further purification. The soluble supernatant was purified by His-tag
purification according to the manufacturer’s protocol. The proteins were loaded onto a HisTrap
FF column, and the column was washed using the elution buffer (50 mmol/L Tris-HCl buffer
containing 250 mmol/L imidazole, 150 mmol/L NaCl). The resulting eluate was dialyzed
overnight against 1000 mL of 50 mmol/L Tris-HCl buffer. The purity of the sample was
determined by SDS-PAGE.
Molecular Docking
The crystal structure of AvPAL from Anabaena variabilis (PBD ID: 2NYN) was used as
template, and the Asn 347 was replaced by Ala to generate the structure of mutant AvPAL-

Appl Biochem Biotechnol
N347A with the maximum overlap; the RMSD (mean square deviation) was less than 2 Å.
Docking of t-CA into the active pockets of AvPAL-N347A and PAL was performed using
Autodock4.2. The t-CA as ligand was downloaded from the PubChem database on NCBI. A
grid-centered box was set to a binding pocket to accommodate the ligand, and the grid-
centered box consisted of conserved amino acids (N223, N347, R317, MIO, Y78, N451,
E448, and Q451) to provide enough space for rotational movement of the ligand. The
Lamarckian genetic algorithm was used in the program. The selection of docking poses was
based on the following criteria: the carboxylate of t-CA should be placed in the corresponding
binding pocket; the carboxylate group of t-CA should form salt a bond with the highly
conserved Arg325, and the MIO and 78Tyr should point toward C and C of the ligand.
α
β
The results were visualized using PyMol.
The Amination Reaction Using AvPAL-N347A
The pure AvPAL-N347A (10 U) and trans-cinnamic acid (10 mmol/L) were added into Tris-HCl
buffer (50 mmol/L, pH 7–11) containing 1–6 mol/L NH OH, and the reaction was shaken at
4
200 r/min at 37 °C for 12 h, and then, 0.5 mL of the sample was extracted for analysis by HPLC.
The Oxidation Reaction Using PmLAAD
The pure PmLAAD (10 U) and DL-phenylalanine (10 mmol/L) were added into Tris-HCl
buffer (50 mmol/L, pH 7–11), containing 1–6 mol/L NH OH; the reaction was carried out in a
4
similar manner to the amination condition.
+
The Effect of NH4 on Stability of PmLAAD
The PmLAAD (10 U) was incubate in Tris-HCl buffer (50 mM, pH 8.5) containing 1–8 mol/L
+
NH4 and 10 mM DL-phenylalanine at 30 °C for 12 h; 0.5 mL of the sample was extracted to
detect the relative activity using HPLC.
The Effect of Reducing Agent NH BH on the Cascade Conversion Using
3
3
AvPAL-N347A and PmLAAD
The effect of the reducing agent NH BH on the cascade conversion using AvPAL-N347A and
3
3
PmLAAD was assayed in Tris-HCl buffer (50 mmol/L, pH 8.5) containing 10 U AvPAL,
0 mmol/L t-CA and 4 mol/L NH OH. The reaction was incubated for 12 h at 35 °C, the t-CA
1
4
was aminated to racemic phenylalanine. Then, 10–60 mmol/L NH BH and PmLAAD (10 U)
3
3
were added to continue the reaction for 12 h. The L-phenylalanine was converted D-phenyl-
alanine through oxidation by PmLAAD and reduction by NH BH . The samples were
3
3
analyzed every 2 h using HPLC.
One-Pot Synthesis of D-Arylalanines
The substituted trans-cinnamic acids (a → n) were used as substrates to synthesize the D-
arylalanines in reaction containing 40 mmol/L NH BH , 4 mol/L NH OH, 10 U AvAL-
3
3
4
N347A, and PmLAAD. In this process, the AvAL-N347A was first added into the reaction
synthesis for 12 h, and then, the NH BH and PmLAAD were added to continue the reaction
3
3

Appl Biochem Biotechnol
for 12 h. The one-pot synthetic reaction was carried out at 35 °C, pH 8.5. The sample was
extracted to analyze the conversion rate and ee of D-arylalanines using HPLC.
D
Activity Assays of AvPAL and PmLAAD
The enzymatic activity of AvPAL was assayed in a reaction mixture (0.5 mL) containing
2
25 μL of Tris-HCl buffer (25 mM, pH 8.5), 250 μL of m-nitro-t-CA (10 mM), and an
appropriate quantity of the enzyme. The reaction was carried out at 35 °C for 1 h and stopped
by the addition of 0.5 mL methanol. The formation of m-nitro-Phe was measured by HPLC.
One enzyme activity unit was defined as the amount of enzyme that produced 1 mmol m-nitro-
Phe per hour at 35 °C. The activity of PmLAAD was performed according to the method
described by Baek [29].
HPLC Analysis
The D-enantiomers and L-enantiomers were detected by HPLC on a C18 column (4.6 mm ×
7
5 mm, Hitachi, Japan) at 210 nm according to the method described by Fukuhara [30]. The
mobile phase contained 20% (v/v) methanol and a complex of optically active L-Pro-Cu(II)
1.5 mmol/L L-Pro and 0.75 mmol/L CuSO ). The retention times of D-enantiomers and L-
(
4
enantiomers are shown in Table S1 (Electronic Supplementary Material), respectively. The eeD
of D-enantiomers was calculated by the following equation:
Dphe−Lphe
Dphe þ Lphe
eeD ð%Þ ¼
 100
where eeD is the enantiomeric excess of D-enantiomers, Lphe, is the concentration of L-
enantiomers; Dphe is the concentration of D-enantiomers.
Results and Discussion
Selection of Mutation Sites Based on Analysis of Structure of AvPAL
PAL and PAM are belonged to MIO-dependent enzyme family, which acts as similar mech-
anism. The TcPAMs from Taxus chinensis and PaPAM from Pantoea agglomerans exhibit
different stereoselectivity and produce R-β-phenylalanine and S-β-phenylalanine, respectively
[31, 32]. The TcPAM catalyzes the isomerization of S-α-phenylalanine to R-β-phenylalanine
through exchanging the position of the amine group (C → C ) and pro-3S hydrogen proton
α
β
(
C → C ) with retention of the configuration at the reaction termini [33], which requires
β α
reorientation after deamination of S-α-phenylalanine to t-CA in which the re-face of the Cβ
and the si-face of the C carton atoms are positioned for amine re-addition and re-protonation
α
[
33, 34]. To achieve the retention of the configuration, the C –C and C –C
of t-CA are
1
a
β
ipso
demonstrated to rotate 180° prior to the rebound of the amino group to C and the hydrogen
β
proton to C . In order to produce D-phenylalanine using AvPAL, the C –C and C –C of t-
α
1
a
β
ipso
CA in active site might be necessary to rotate. Therefore, the binding orientation of t-CA in
active site was resolved using molecular docking. According to the structure and results of
docking (Fig. 1), the carboxylate binding pocket in the active site consists of three highly
conserved Asn (N233, N347, N451) residues and an Arg residue at position 317, the

Appl Biochem Biotechnol
Fig. 1 The binding mode in active site between trans-cinnamic acid with active site. The substrate trans-
cinnamic acid (t-CA, blue) was docked into WtPAL and mutant AvPAL-N347A, in which two binding
orientations were found. a The MIO-NH
2
a β
adducts and Tyr78 point to Si-face of C and Re-face of C of t-
CA; c MIO-NH adducts and Tyr78 point to Re-face of C
2
a
and Si-face of C of t-CA. b, d The hydrogen bond
β
network of two binding orientations between residues in active site with t-CA. The hydrogen bond network was
drawn using ChemDraw according to the a, c part in this figure. The hydrogen bond and salt bridge were showed
as black and yellow dotted lines, respectively. The key residues in active site are colored by atom as follows: C
(
green), O (red), and N (blue)
carboxylate group of t-CA forms a salt bond with highly conserved Arg317, and the three Asn
residues are involved in formation of a hydrogen bond network. The hydrogen bond network
might control the accommodation of t-CA with different binding orientations. As shown in
Fig. 1a, b, the si-face of C points toward the MIO-NH adduct and the 78Tyr-H proton points
a
2
toward the re-face of Cβ; this binding mode prefers to produce L-phe. To produce D-phe,
the binding orientation needs to be switched to point the re-face of C toward the MIO-
a
NH adduct and the 78Tyr-H proton toward the si-face of C , as shown in Fig. 1c, d. To
2
β
achieve the switch in binding modes, the C –Cα and C –C bond were considered to
ipso
1
β
rotate to expose the re-face to the MIO-NH and the si-face to the 78Tyr-H proton, which
2
would be constricted by the key residue N347 in the hydrogen bond network. Therefore,
the N347 was mutated to Ala to perturb the hydrogen bond network and facilitate the
rotation to improve D-selectivity.

Appl Biochem Biotechnol
Effects of Mutation at Residue 347 Site on Stereoselectivity
The mutant and wild-type genes were expressed in E. coli BL21 (DE3). After purification, the
mutant AvPAL-N347A and wild-type AvPAL (WtPAL) proteins appeared as a single band of
approximately 50 kDa on SDS-PAGE (Fig. 2), in agreement with the molecular weight
calculated from the amino acid sequence. Consequently, the mutant AvPAL-N347A exhibits
high stereoselectivity for D-m-nitro-phe. As shown in Fig. 3, the activities of WtPAL for L-m-
nitro-phe and D-m-nitro-phe were 5.6 and 3.1 U/mg, respectively. However, the activity of
mutant AvPAL-N347A for D-m-nitro-phe was 2.3-fold higher (7.3 U/mg) than WtPAL. Al-
though, the activity for D-selectivity of AvPAL-N347A is lower than that of mutants H359Y
(11.35 U/mg) and H359K (10.76 U/mg) [26], the N347 is another key amino acid to
manipulate the stereoselectivity. Furthermore, CD was used to investigate the structures of
WtPAL and mutant AvPAL-N347A. The results showed that the increased activity for D-m-
nitro-phe might result from the perturbance of the hydrogen bond network and improved the
rotation of the C –C bond and not change the structure. The CD spectrum of the mutant was
1
α
similar to that of the wild type (Fig. 4).
The Synthesis Route of D-Arylalanine
Although the activity of AvPAL-N347A for D-m-nitro-phe was increased, its activity for L-m-
nitro-phe remained at 2.4 U/mg, and the product is still D,L-mixture. To obtain highly pure D-
enantiomer, it was necessary to convert the L-enantiomer to D-enantiomer. Therefore, the
synthesis route was referred to previous reports [26, 35], which is shown in Scheme 1. The
L-enantiomer product was transformed to the intermediate amino acid through oxidation by
PmLAAD [28], and then, the amino acid was reduced to D-enantiomer and L-enantiomer by
Fig. 2 SDS-PAGE of recombinant enzyme expressed in E. coli BL21. M, molecular weight marker; a1, the cell
extracts of E.coli BL21 (DE3) harboring pET28a-laad; a2, the purified PmLAAD; b1, the cell extracts of E. coli
BL21 (DE3) harboring pET28a-pal; b2, the purified AvPAL; b3, the cell extracts of E. coli BL21 (DE3)
harboring pET28a-pal-N347A; b4, the purified AvPAL-N347A

Appl Biochem Biotechnol
Fig. 3 The activity of AvPAL and
mutant AvPAL-N247A. The
activities for L-m-nitro-phe and D-
m-nitro-phe were signified in gray
and rosiness
amine borane (NH BH ) [35], the D-enantiomer was continually accumulated in reaction.
3
3
However, in the cascade process of Parmeggiani [26, 35], the PAL, LAAD, and NH BH were
3
3
simultaneously added into reaction system, the result is that only t-CAs with electron-
withdrawing groups (such as nitro, fluoro) can be converted to corresponding D-product in
the presence of NH BH , and the t-CA or t-CAs with electron-donating group (such as methyl,
3
3
hydroxyl) in phenyl ring could not be converted in the presence of NH BH , which might
3
3
result from the deactivation of AvPAL by NH BH . In this work, the cascade process was
3
3
modified, which the AvPAL, PmLAAD, and NH BH were successively added into the
3
3
reaction system to resolve the deactivation of AvPAL by NH BH and convert the t-CA and
3
3
its derivatives with electron-donating groups (such as methyl and hydroxyl).Thus, the D-
enantiomer could be constantly accumulated.
The PmLAAD was prepared through heterologous expression. In order to achieve soluble
expression in E. coli, the gene sequence of laad from P. mirabilis was modified by removing
one N-terminal transmembrane region, the methods was referred to the references [27, 28].The
open reading frame of the PmLAAD gene is 1350 bp long, encoding 450 amino acids. The
recombinant expression vector pET-28a-laad was constructed and then transformed into
E. coli BL21 (DE3) for PmLAAD expression. SDS-PAGE analysis showed that the recom-
binant PmLAAD polypeptide was approximately 50 kDa (Fig. 2), which is agreement with the
Fig. 4 The conformation change
of the mutants with circular
dichroism (CD). The CD spectra
were measured at a protein
concentration of 0.1 mg/mL in
50 mM Tris-HCl buffer (pH 8.6)

Appl Biochem Biotechnol
Scheme 1 The synthesis process of D-arylalanines
molecular weight calculated from the amino acid sequence. The recombinant PmLAAD was
purified by His-tag-purification using an AkTA-purifier; PmLAAD appeared as a single band
of approximate 50 kDa on an SDS-PAGE.
The Conditions of AvPAL-N347A-Catalyzed Amination and PmLAAD-Catalyzed
Oxidation
The amination and oxidation conditions are different due to the different enzymatic properties
of the two enzymes. To obtain optimal reaction conditions, the amination and oxidation were
assayed as shown in Fig. 5; the maximal amination rate of trans-cinnamic acid was achieved at
conditions of 4–5 mol/L NH OH, 40–50 °C, pH 9–10, reaching 93% amination. However, the
4
maximal oxidation rate of L-phenylalanine was obtained at conditions of 1–4 mol/L NH OH,
4
3
0–40 °C, and pH 7–8. Therefore, in order to reach a high amination rate and oxidation rate
simultaneously, the optimal reaction conditions were determined as 4 mol/L NH OH, 35 °C,
4
and pH 8.5.
The Effects of Ammonia on Stability of PmLAAD
+
The PmLAAD (10 U) was incubate in solution containing 1–8 mol/L NH4 for 12 h. The
+
4
enzyme was stable at 1–5 mol/L NH , no activity decrease was observed under 1–2 mol/L
+
4
+
4
NH . The activity maintained about 90% at 5 mol/L NH , and about 60% of activity
a
b
c
+
4
Fig. 5 The amination and oxidation conditions using AvPAL-N347A and PmLAAD. The effects of NH
concentration, pH and temperature on amination and oxidation rate, respectively

Appl Biochem Biotechnol
+
4
Fig. 6 Effect of NH
of PmLAAD
on stability
+
remained after incubation at 8 mol/L NH4 (Fig. 6). The result was similar to the previous
+
works, which optimal concentration is 5 mol/L NH4 [26].
The Effect of Reducing Agent NH BH on the Cascade Conversion Using
3
3
AvPAL-N347A and PmLAAD
The conversion reaction was carried out over 24 h at 35 °C, and pH 8.5, the reaction system
consists of 10–60 mmol/L NH BH , 4 mol/L NH OH, and 10 U AvPAL-N347A and
3
3
4
PmLAAD. Samples were analyzed to calculate the conversion of trans-cinnamic acid to D-
phenylalanine. The results are shown in Fig. 7 and the conversion rate increased with the
increasing concentration of NH BH and exceeded 80% at 40 mmol/L NH BH . However, if
3
3
3
3
the concentration of NH BH was further increased, the conversion rate was gradually
3
3
decreased, which might be a result of the inhibitory activity of PmLAAD at high concentra-
tions reduction agent.
Fig. 7 Effect of reducing agent
NH BH on the cascade
3 3
conversion using AvPAL-N347A
and PmLAAD

Appl Biochem Biotechnol
Table 1 The effects of different group at phenyl ring on conversion rate and optical purity
Substrate
R
Conversion (%)
ee
D
(%)
Substrate
R
Conversion (%)
D
ee (%)
a
b
c
d
e
f
H
82
69
91
72
69
96
78
> 99
> 99
> 99
> 99
> 99
> 99
> 99
1 h
k
j
l
m
n
p-Me
m-Me
o-Me
p-OH
m-OH
o-OH
12
23
16
13
nd
26
92
93
89
90
nd
72
p-F
m-F
o-F
p-NO
m-NO
o-NO
2
2
g
2
The ee
D
of D-enantiomer surpassing 99% indicated that the L-enantiomer was not detected using HPLC
, enantiomer excess of D-enantiomer
nd, product was not detected; ee
D
One-Pot Preparation of D-Arylalanines Using AvPAL-N347A Coupling PmLAAD
A series of substituted trans-cinnamic acids (a → n, Table 1) were used as substrates to
synthesize D-arylalanines at conditions of 40 mmol/L NH BH , 4 mol/L NH OH, and 10 U
3
3
4
AvPAL-N347A and PmLAAD. The one-pot synthetic reaction was carried out at 35 °C, pH
.5, and for 16 h. As shown in Table 1, substrates with fluoro, nitro, and methyl substituent
8
groups are accommodated by AvPAL-N347A and PmLAAD; the conversion rate and ee were
D
found to be varied based on the electronic properties and position of the substituent group. The
conversions tend to follow the following pattern: meta-substituted trans-cinnamic acid exhibits
higher total conversion rate than ortho- and para-substituted trans-cinnamic acids. This might
be a result of a narrow and long hydrophobic pocket containing highly conservative residues
(such as 104Leu, 107Phe, 108Leu, 219Leu, 222Met, and 448E) (Fig. 8), in which it might
exist close to the para and ortho position and the width might be insufficient to accommodate
the ortho- and para-substituent substrates. The conversion was also found to be dominated by
the electronic properties of ring-substituent group. The trans-cinnamic acids with strongly
electron-withdrawing groups (such as nitro) at the phenyl ring are converted predominantly to
D-arylalanines. This was especially, true of trans-cinnamic acid with the m-nitro group, which
was transferred into 96% of D-product, and the ee exceeded 99%. The results are similar to
D
the previous works, and the highest conversion rate was 80% (ee of 98%) [26]. In contrast
D
with the process of Parmeggiani [26], the t-CAwith strongly electron-donating groups (such as
Fig. 8 Phenyl ring binding pocket of AvPAL. The binding pocket for the aromatic ring of t-CA is shown in
orange and consists of hydrophobic residues (such as 104Leu, 107Phe, 108Leu, 219Leu, 222Met). The salt
bridge between 317Arg with carboxylate group of t-CA is shown as black dotted lines. The oxygen, nitrogen, and
carbon atoms are shown in red, blue, and gray, respectively

Appl Biochem Biotechnol
methyl or hydroxyl) could be converted in spite of low conversion rate (Table 1), which might
result from low activity of AvPAL for substrate with electron-donating groups. Therefore, the
AvPAL is needed to further enhance activity for t-CA with electron-donating group in phenyl
ring using molecular modification and expand the application in synthesis of D-arylalanines.
Conclusions
In this paper, we successfully generated a mutant PAL with high stereoselectivity for D-m-
nitro-phe through molecular modification based on structure and mechanism. The
stereoselectivity of the mutant for D-m-nitro-phe is approximately 2.3-fold higher than that
of WtPAL. The AvPAL-N347A from A. variabilis exhibits activity toward a broad substrate
scope, especially toward m-F and m-NO . The PmLAAD from P. mirabilis showed high
2
selectivity for aromatic amino acids. Using the AvPAL-N347A-catalyzed animation and
PmLAAD-catalyzed oxidation, one-pot synthetic process for D-arylalanines was successfully
constructed using t-CA and its derivatives as substrates. The conversion rate of t-CA reached
8
2% and ee exceeded 99%. The t-CA with the electron-withdrawing nitro group at the meta-
D
position proved to be the most suitable substrate, and the conversion rate reached 96% and eeD
exceeded 99%. Moreover, the t-CA with strongly electron-donating groups (such as methyl or
hydroxyl) could be converted in spite of low conversion rate and the conversion rate of
substrate with m-Me reached 23%. In contrast with the hydantoinase method and previous
cascade process, this modified one-pot enzyme synthesis using AvPAL-N347A, PmLAAD,
and NH BH afforded an alternative approach for synthesizing highly pure D-arylalanines.
3
3
Acknowledgments The authors gratefully acknowledged Prof. Zheming Zhou from the School of Biotechnol-
ogy, Jiangnan University for the facilities and infrastructure. The authors also acknowledged the help rendered by
Weifeng Sun from Xihua University in proofreading the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (21506172;
31671797), Natural Sciences Foundation supported by Anhui Province universities (KJ2016A801) and Anhui
Polytechnic University Youth Talent Support Program (2016BJRC006).
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
References
1
2
. Genchi, G. (2017). An overview on D-amino acids. Amino Acids, 49(9), 1521–1533.
. Parmeggiani, F., Ahmed, S. T., Thompson, M. P., Weise, N. J., Galman, J. L., Gahloth, D.,
Dunstan, M. S., Leys, D., & Turner, N. J. (2016). Single-biocatalyst synthesis of enantiopure D-
arylalanines exploiting an engineered D-amino acid dehydrogenase. Advanced Synthesis and
Catalysis, 358(20), 3298–3306.
3
. Fox, M. E., Jackson, M., Meek, G., & Willets, M. (2011). Large-scale synthesis of a substituted D-phenylalanine
using asymmetric hydrogenation. Organic Process Research and Development, 15(5), 1163–1171.
. Muller, U., van Assema, F., Gunsior, M., Orf, S., Kremer, S., Schipper, D., Wagemans, A., Townsend, C.
A., Sonke, T., & Bovenberg, R. (2006). Metabolic engineering of the E. coli L-phenylalanine pathway for
the production of D-phenylglycine (D-Phg). Metabolic Engineering, 8(3), 196–208.
4

Appl Biochem Biotechnol
5. Breuer, M., Ditrich, K., Habicher, T., Hauer, B., Kesseler, M., Sturmer, R., & Zelinski, T. (2004). Industrial
methods for the production of optically active intermediates. Angewandte Chemie, International Edition,
43(7), 788–824.
6
. Kobayashi, J., Shimizu, Y., Mutaguchi, Y., Doi, K., & Ohshima, T. (2013). Characterization of D-amino acid
aminotransferase from Lactobacillus salivarius. Journal of Molecular Catalysis B Enzymatic, 94, 15–22.
. Liu, R. X., Liu, S. P., Cheng, S., Zhang, L., Ding, Z. Y., Gu, Z. H., & Shi, G. Y. (2015). Screening,
characterization and utilization of D-amino acid aminotransferase to obtain D-phenylalanine. Applied
Biochemistry and Microbiology, 51(6), 695–703.
7
8. Hossain, G. S., Li, J., Shin, H., Du, G., Liu, L., & Chen, J. (2014). L-Amino acid oxidases from microbial
sources: types, properties, functions, and applications. Applied Microbiology and Biotechnology, 98(4),
1507–1515.
9. Singh, S., Gogoi, B. K., & Bezbaruah, R. L. (2011). Racemic resolution of some DL-amino acids using
Aspergillus fumigatus L-amino acid oxidase. Current Microbiology, 63(1), 94–99.
1
0. Park, J. H., Kim, G. J., & Kim, H. S. (2000). Production of D-amino acid using whole cells of recombinant
Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase. Biotechnology
Progress, 16(4), 564–570.
1
1. Altenbuchner, J., Siemann-Herzberg, M., & Syldatk, C. (2001). Hydantoinases and related enzymes as
biocatalysts for the synthesis of unnatural chiral amino acids. Current Opinion in Biotechnology, 12(6),
59–563.
5
1
1
1
2. Gao, X. Z., Ma, Q. Y., & Zhu, H. L. (2015). Distribution, industrial applications, and enzymatic synthesis of
D-amino acids. Applied Microbiology and Biotechnology, 99(8), 3341–3349.
3. Foster, I. M., Dorrington, R. D., & Burton, S. G. (2003). Enhanced hydantoinase and N-carbamoylase
activity on immobilisation of Agrobacterium tumefaciens. Biotechnology Letters, 25(1), 67–72.
4. Parmeggiani, F., Ahmed, S. T., Weise, N. J., & Turner, N. J. (2016). Telescopic one-pot condensation-
hydroamination strategy for the synthesis of optically pure L-phenylalanines from benzaldehydes.
Tetrahedron, 72(46), 7256–7262.
1
1
1
1
1
2
2
2
5. Sanchez-Murcia, P. A., Bueren-Calabuig, J. A., Camacho-Artacho, M., Cortes-Cabrera, A., & Gago, F.
(2016). Stepwise simulation of 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) biogenesis in histi-
dine ammonia-lyase. Biochemistry, 55(41), 5854–5864.
6. Watts, K. T., Mijts, B. N., Lee, P. C., Manning, A. J., & Schmidt-Dannert, C. (2006). Discovery of a
substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family.
Chemistry & Biology, 13(12), 1317–1326.
7. Heberling, M. M., Masman, M. F., Bartsch, S., Wybenga, G. G., Dijkstra, B. W., Marrink, S. J., & Janssen,
D. B. (2015). Ironing out their differences: dissecting the structural determinants of a phenylalanine
aminomutase and ammonia lyase. ACS Chemical Biology, 10(4), 989–997.
8. Walter, T., King, Z., & Walker, K. D. (2016). A tyrosine aminomutase from rice (Oryza sativa) isomerizes
(
S)-alpha- to (R)-beta-tyrosine with unique high enantioselectivity and retention of configuration.
Biochemistry, 55(1), 1–4.
9. Lovelock, S. L., Lloyd, R. C., & Turner, N. J. (2014). Phenylalanine ammonia lyase catalyzed synthesis of
amino acids by an MIO-cofactor independent pathway. Angewandte Chemie, International Edition, 53(18),
4652–4656.
0. Pinto, G. P., Ribeiro, A. J. M., Ramos, M. J., Fernandes, P. A., Toscano, M., & Russo, N. (2015). New
insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models.
Archives of Biochemistry and Biophysics, 582, 107–115.
1. Bencze, L. C., Filip, A., Banoczi, G., Tosa, M. I., Irimie, F. D., Gellert, A., Poppe, L., & Paizs, C. (2017).
Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards
styrylalanines. Organic & Biomolecular Chemistry, 15(17), 3717–3727.
2. Jaliani, H. Z., Farajnia, S., Mohammadi, S. A., Barzegar, A., & Talebi, S. (2013). Engineering and kinetic
stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase. Applied
Biochemistry and Biotechnology, 171(7), 1805–1818.
2
3. Lovelock, S. L., & Turner, N. J. (2014). Bacterial Anabaena variabilis phenylalanine ammonia lyase: A
biocatalyst with broad substrate specificity. Bioorganic and Medicinal Chemistry, 22(20), 5555–5557.
4. DreSsen, A., Hilberath, T., Mackfeld, U., Billmeier, A., Rudat, J., & Pohl, M. (2017). Phenylalanine ammonia
lyase from Arabidopsis thaliana (AtPAL2): a potent MIO-enzyme for the synthesis of non-canonical aromatic
alpha-amino acids. Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1)
and Rhodosporidium toruloides (RtPAL). Journal of Biotechnology, 258, 148–157.
2
2
5. DreSsen, A., Hilberath, T., Mackfeld, U., Billmeier, A., Rudat, J., & Pohl, M. (2017). Phenylalanine
ammonia lyase from Arabidopsis thaliana (AtPAL2): a potent MIO-enzyme for the synthesis of non-
canonical aromatic alpha-amino acids. Part II: Application in different reactor concepts for the production of
(S)-2-chloro-phenylalanine. Journal of Biotechnology, 258, 158–166.

Appl Biochem Biotechnol
2
2
2
2
6. Parmeggiani, F., Lovelock, S. L., Weise, N. J., Ahmed, S. T., & Turner, N. J. (2015). Synthesis of D- and L-
phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process.
Angewandte Chemie, International Edition, 54(15), 4608–4611.
7. Liu, L., Hossain, G. S., Shin, H. D., Li, J. H., Du, G. C., & Chen, J. (2013). One-step production of alpha-
ketoglutaric acid from glutamic acid with an engineered L-amino acid deaminase from Proteus mirabilis.
Journal of Biotechnology, 164(1), 97–104.
8. Motta, P., Molla, G., Pollegioni, L., & Nardini, M. (2016). Structure-function relationships in L-amino acid
deaminase, a flavor protein belonging to a novel class of biotechnologically relevant enzymes. The Journal
of Biological Chemistry, 291(20), 10457–10475.
9. Baek, J. O., Seo, J. W., Kwon, O., Seong, S. I., Kim, I. H., & Kim, C. H. (2011). Expression and
characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli.
Journal of Basic Microbiology, 51(2), 129–135.
30. Fukuhara, T. Y. S. (1990). Novel ligand-exchange chromatographic resolution of DL-amino acids using
nucleotides and coenzymes. Journal of Chromatographic Science, 28(1), 114–117.
3
1. Wybenga, G. G., Szymanski, W., Wu, B., Feringa, B. L., Janssen, D. B., & Dijkstra, B. W. (2014).
Structural investigations into the stereochemistry and activity of a phenylalanine-2,3-aminomutase from
Taxus chinensis. Biochemistry, 53(19), 3187–3198.
32. Ratnayake, N. D., Wanninayake, U., Geiger, J. H., & Walker, K. D. (2011). Stereochemistry and mechanism
of a microbial phenylalanine aminomutase. Journal of the American Chemical Society, 133(22), 8531–
8533.
33. Mutatu, W., Klettke, K. L., Foster, C., & Walker, K. D. (2007). Unusual mechanism for an aminomutase
rearrangement: retention of configuration at the migration termini. Biochemistry, 46(34), 9785–9794.
34. Feng, L., Wanninayake, U., Strom, S., Geiger, J., & Walker, K. D. (2011). Mechanistic, mutational, and
structural evaluation of a Taxus phenylalanine aminomutase. Biochemistry, 50(14), 2919–2930.
35. Alexandre, F. R., Pantaleone, D. P., Taylor, P. P., Fotheringham, I. G., Ager, D. J., & Turner, N. J. (2002).
Amine-boranes: effective reducing agents for the deracemisation of DL-amino acids using L-amino acid
oxidase from Proteus myxofaciens. Tetrahedron Letters, 43(4), 707–710.