Covalent Linkage of an R-ω-Transaminase to a d-Amino Acid Oxidase through Protein Splicing to Enhance Enzymatic Catalysis of Transamination

Article abstract of DOI:10.1002/cbic.201800646

R-ω-Transaminases (RTAs) catalyse the conversion of R-configured amines [e.g., (R)-1-phenylethylamine] into the corresponding ketones (e.g., acetophenone), by transferring an amino group from an amino donor [e.g., (R)-1-phenylethylamine] onto an amino acceptor (e.g., pyruvate), resulting in a co-product (e.g., d-alanine). d-Alanine can be deaminated back to pyruvate by d-amino acid oxidase (DAAOs). Here, through in vivo subunit splicing, the N terminus of an RTA subunit (RTA^S) was specifically ligated to the C terminus of a DAAO subunit (DAAO^S) through native peptide bonds (RTA&DAAO). RTA^S is in close proximity to DAAO^S, at a molecular-scale distance. Thus the transfer of pyruvate and d-alanine between RTA and DAAO can be directional and efficient. Pyruvate→d-alanine→pyruvate cycles are efficiently formed, thus promoting the forward transamination reaction. In a different, in vitro noncovalent approach, based on coiled-coil association, the RTA^S N terminus was specifically associated with the DAAO^S C terminus (RTA#DAAO). In addition, the two mixed individual enzymes (RTA+DAAO) were also studied. RTA&DAAO has a shorter distance between the paired subunits (RTA^S–DAAO^S) than RTA#DAAO, and the number of the paired subunits is higher than in the case of RTA#DAAO, whereas RTA+DAAO cannot form the paired subunits. RTA&DAAO exhibited a transamination catalysis efficiency higher than that of RTA#DAAO and much higher than that of RTA+DAAO.

Full text of DOI:10.1002/cbic.201800646

Accepted Article
Title: R-ɷ-Transaminase Covalently Linking to D-Amino Acid Oxidase
Through Protein Splicing to Enhance Enzymatic Catalysis of
Transamination of Amines
Authors: Wei Feng, Kun Du, Rong Li, and Dongrui Zhang
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201800646
Link to VoR: http://dx.doi.org/10.1002/cbic.201800646
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ChemBioChem
10.1002/cbic.201800646
R-ɷ-Transaminase Covalently Linking to D-Amino Acid Oxidase
Through Protein Splicing to Enhance Enzymatic Catalysis of
Transamination of Amines
†
†
*
Kun Du , Rong Li , Dongrui Zhang, Wei Feng
R-ɷ-Transaminases (RTA) catalyze the conversion of (R)-amines (e.g. (R)-1-
phenylethylamine) to corresponding ketones (e.g. acetylbenzene), by transferring an
amino group from an amino donor (e.g. (R)-1-phenylethylamine) onto an amino
acceptor (e.g. pyruvate), resulting in a co-product (e.g. D-alanine). D-alanine can be
deaminated back to pyruvate by D-amino acid oxidase (DAAO). Herein, through the
S
vivo subunit splicing, the N-terminus of the RTA subunit (RTA ) was specifically
S
ligated to the C-terminus of the DAAO subunit (DAAO ) with native peptide bonds
S
S
(
RTA&DAAO). RTA is in close proximity to DAAO at a molecular distance. Thus
the transferring of pyruvate and D-alanine between RTA and DAAO can be directional
and efficient. The pyruvateD-alaninepyruvate cycles are efficiently formed,
promoting the transamination reaction forward. In an in vitro non-covalent approach,
S
through the coiled-coil association, the N-terminus of RTA was specifically associated
S
with the C-terminus of DAAO (RTA#DAAO). In addition, the mixed two enzymes
RTA+DAAO was also studied. RTA&DAAO has a shorter distance between the paired
S
S
subunits RTA DAAO than RTA#DAAO, and the number of the paired subunits is
higher than that of RTA#DAAO. While RTA+DAAO cannot form the paired subunits.
For transamination of the amines, RTA&DAAO exhibited a higher catalysis efficiency
*
Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, China.
E-mail: fengwei@mail.buct.edu.cn
These authors contributed equally to this work
†
1
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ChemBioChem
10.1002/cbic.201800646
than RTA#DAAO, and a much higher catalysis efficiency than RTA+DAAO.
Keywords: R-ɷ-Transaminase; D-amino acid oxidase; protein splicing; pyruvate
Introduction
Chiral amines are important chemicals for the synthesis of pharmaceuticals.[1,2,3] ɷ-
[
4]
Transaminases (ɷ-TAs) are multimeric enzymes and have been investigated for the
[5]
preparation of chiral amines. The amines in optically pure form can be used for the
synthesis of biologically active compounds.[6] (R)-enantioselective transaminases
[
7]
(
RTAs) have been used for the kinetic resolution of racemic amines. RTAs catalyze
the conversion of (R)-amines (e.g. (R)-1-phenylethylamine) to corresponding ketones
e.g. acetylbenzene), by transferring an amino group from an amino donor (e.g. (R)-1-
phenylethylamine) onto an amino acceptor (e.g. pyruvate), resulting in a co-product
e.g. D-alanine). Pyruvate has been extensively used as amino acceptor in RTA
(
(
[8]
catalyzing processes. Catalytic amount of pyruvate favors the transamination reaction
[
8]
and facilitates subsequent purification. This requires the recycling of pyruvate in situ.
D-amino acid oxidases (DAAOs) are dimeric enzymes that catalyze a D-amino acid
substrate into α-keto acid.[9] D-amino acid oxidase was used together with (R)-
enantioselective transaminase,[10] catalyzing the deamination of D-alanine back to
pyruvate, achieving the recycling of pyruvate in situ.
However, for the conversion of some amines, for example (R)-phenethylamine, (R)-
1
-aminoindan, and furfurylamine, we found that the combined two enzymes
RTA+DAAO achieved a very low substrate conversion. It is speculated that D-alanine
and pyruvate were transferred between RTA and DAAO with a low efficiency. Spatially
arranging RTA and DAAO at a short distance is a solution to improving the transport
efficiency. It is ideal that the distance between RTA and DAAO is at a molecular
distance. This requires the RTA and DAAO subunits being arranged in a one-by-one
2
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ChemBioChem
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fashion. Gene fusion can be a solution to linking two enzymes.[11] We have fused the
genes of RTA and DAAO, and the fusion gene was expressed in Escherichia coli.
However, the expressed fusion enzyme did not exhibit any enzymatic activity. Possibly,
it is because the multimeric enzymes RTA and DAAO having large molecular weights,
the fusion enzyme did not have a correct folding in the Escherichia coli cell.
Previously, we have specifically ligated two multimeric enzymes with native
peptides through intein-mediated in vivo subunit splicing.[12,13] Herein, we extend the
in vivo subunit splicing methodology to specifically ligate demeric RTA and dimeric
DAAO, in order to investigate the importance of efficiently forming the pyruvate D-
alanine pyruvate cycles between RTA and DAAO for transamination reaction. The
C-terminal domain of the split NpuDnaE intein [14] is fused to the N-terminus of the
RTA subunit. The N-terminal domain of the split NpuDnaE intein is fused to the C-
terminus of the DAAO subunit. Through the intein-mediated subunit splicing,[12] the
N-terminus of the RTA subunit is specifically ligated to the C-terminus of the DAAO
subunit with native peptides, as illustrated in Figure 1. Thus, the RTA and DAAO
subunits can be spatially arranged in a one-by-one fashion at a molecular distance. For
comparison, an in vitro non-covalent approach is investigated. An arginine-rich leucine
zipper (Z
R
) [15] is fused to the N-terminus of RTA subunit, and a glutamic acid-rich
) [15] is fused to the C-terminus of DAAO subunits. The N-terminus
leucine zipper (Z
E
of RTA subunit is associated with the C-terminus of DAAO subunit through the coiled-
coil association of Z and Z in vitro, resulting in an association product RTA#DAAO,
E
R
as illustrated in Figure 2. The two-enzyme systems have been evaluated for the
conversion of amines. Their stability is also investigated.
Results
Two-enzyme system RTA&DAAO though in vivo subunit splicing
3
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ChemBioChem
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R-ɷ-Transaminase (RTA) and D-Amino oxidase (DAAO) are dimeric enzymes. The N-
terminal domain of the split NpuDnaE intein (IntN) [14] was fused to the C-terminus of
C [14]
the DAAO subunit. The C-terminal domain of the split NpuDnaE intein (Int ) was
fused to the N-terminus of the RTA subunit (supplementary Figure S1). A rigid α-
C
helical linker (EPPPPLPPPPLPPPPEPPPP) between Int and the RTA subunit and
N
between Int and the DAAO subunit are required, in order to reduce the steric hindrance
for the splicing of multimeric enzymes.
The splicing product RTA&DAAO was purified through Ni Sepharose beads and
ultrafiltration fractionation. Thus those RTA and DAAO, that were not involved in the
splicing reactions, were separated from the splicing product RTA&DAAO. The
purified RTA&DAAO was analyzed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) (Figure 3a). The top band is ascribed to the RTAS-
S
S
DAAO subunit. It is the key subunit in RTA&DAAO. Appearance of the RTA -
S
DAAO subunit band confirmed the ligation of the RTA and DAAO subunits through
C
N
the in vivo posttranslational subunit splicing. The bands for Int -link-RTA and Int -
link-DAAO are ascribed to those subunits that did not take part in the subunit splicing
S
S
reaction, but they were associated with the RTA -DAAO subunit in RTA&DAAO
(
(
Figure 3c). In comparison to the RTA (supplementary Figure S2a) and DAAO
C
N
supplementary Figure S2b) subunits, the Int -link-RTAand Int -link-DAAO subunits
exhibited larger molecular weights due to being fused to the C- and N-terminal domains
of the split intein, respectively.
RTA&DAAO was analyzed by MALDI-TOF-MS in order to determine its molecular
weight. However, the measured data are not reliable, possibly because the
RTA&DAAO fractions have large molecular weights. RTA&DAAO was analyzed by
blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE has been often
4
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ChemBioChem
10.1002/cbic.201800646
used for identification of proteins from multiprotein complexes.[16] The BN-PAGE of
RTA&DAAO is illustrated in Figure 3b. Six bands appeared for RTA&DAAO. Lane
M is for Marker proteins. Based on lane M, the relationship of the electrophoretic
mobility of marker proteins versus the molecular weight was obtained, as shown in
supplementary Figure S3a. The function shown in Figure S3a was used to estimate the
apparent molecular weights (AMWs) corresponding to the six bands, by comparing the
electrophoretic mobility of the bands with that of marker proteins. The AMWs of the
bands I, II, III, IV, V, and VI were determined to be 1152, 710, 581, 522, 399, and 268
kDa, respectively. Then we searched the ligation modes with theoretical molecular
weights (TMWs) being in accordance with these AMWs. Based on the molecular
C
N
weights of intein and the Int -link-RTA and DAAO-link-Int subunits, the TMWs of
the possible ligation modes were predicated. Supplementary Table S1 lists those
ligation modes with TMWs being in accordance with the AMWs of the six bands. The
ligation modes were finally determined to be, RTA~DAAO~RTA~DAAO~RTA~
DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA (band I);
RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA (band II); DAAO~
RTA~DAAO~RTA~DAAO~RTA~DAAO (band III); RTA~DAAO~RTA~DAAO~
RTA~DAAO~RTA (band IV); RTA~DAAO~RTA~DAAO~RTA (band V); DAAO~
RTA~DAAO (band VI). Figure 3c schematically illustrates the ligation modes
S
S
corresponding to the six bands. The RTA -DAAO subunit (as illustrated by
in
Figure 3c) is formed by the hetrosubunit ligation with a peptide bond through the
subunit splicing, and it is the key subunit in the RTA&DAAO fractions. Those RTA
and DAAO subunits that are not involved in the subunit splicing are associated with the
S
S
RTA -DAAO subunit, as illustrated in Figure 1. The relative abundances of the six
bands were estimated based on the intensity, they are 10.1%, 11.7%, 0.5%, 1.0%, 7.5%,
5
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ChemBioChem
10.1002/cbic.201800646
and 69.2% for bands I, II, III, IV, V, and VI, respectively. The averaged apparent
molecular weight of RTA&DAAO was estimated to be 408 kDa
Two-enzyme system RTA#DAAO though in vitro subunit association
In an in vitro non-covalent approach, an arginine-rich leucine zipper (Z
the N-terminus of the RTA subunit, and a glutamic acid-rich leucine zipper (Z
fused to the C-terminus of the DAAO subunit (supplementary Figure S4). The rigid α-
helical linker (EPPPPLPPPPLPPPPEPPPP) between Z and the RTA subunit and
between Z and the DAAO subunit are required in order to promote the coiled-coil
association of Z and Z . The purified DAAO-Z and RTA-Z were analyzed by SDS-
PAGE (supplementary Figure S5). The N-terminus of the RTA subunit was associated
with the C-terminus of the DAAO subunit through the coiled-coil association of Z and
, resulting in the association product RTA#DAAO, and the RTA =DAAO subunit
as illustrated in Figure 2) is the key subunit. The BN-PAGE analysis (Figure 4a)
revealed that RTA#DAAO comprises three fractions. The apparent molecular weights
AMWs) corresponding to the three bands were estimated by comparing the
R
) was fused to
E
) was
R
E
R
E
E
R
E
S
S
Z
R
(
(
electrophoretic mobility of the bands with that of marker proteins (supplementary
Figure S3b). The AMWs of the bands I, II, and III are 560, 419, and 289 kDa,
respectively. Then we searched the association modes with theoretical molecular
weights (TMWs) being in accordance with these AMWs. Based on the molecular
weights of the RTA-Z
R
and DAAO-Z subunits, the theoretical molecular weights of
E
possible association modes were predicated. Supplementary Table S2 lists those
association modes with TMWs being in accordance with the AMWs of the three bands.
The
association
modes
were
finally
determined
to
be
RTA≈DAAO≈RTA≈DAAO≈RTA≈DAAO≈RTA (band I); RTA≈DAAO≈RTA≈
DAAO≈RTA (band II); DAAO≈RTA≈DAAO (band III). (Figure 4b). The relative
6
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ChemBioChem
10.1002/cbic.201800646
abundances of the bands were estimated based on the intensity, they are 5.4% for band
I, 16.3% for band II, and 78.3% for band III. The apparent molecular weight of
RTA#DAAO was estimated to be 309 k.
S
S
S
Schematic presentations in Figures 3c and 4b illustrate that the RTA -DAAO
S
subunit is the key subunit in the splicing product RTA&DAAO and the RTA =DAAO
subunit is the key subunit in the association product RTA#DAAO. Based on the average
S
S
AMWs of RTA&DAAO and RTA#DAAO and the MWs of the RTA -DAAO and
S
S
S
S
RTA =DAAO subunits, the number of the RTA -DAAO subunit in RTA&DAAO is
S
S
2
3.1% higher than that of the RTA =DAAO subunit in RTA#DAAO. It means that
RTA&DAAO comprises more paired subunits than RTA#DAAO.
Stability of RTA&DAAO and RTA#DAAO
The stability of multimeric enzymes is also of significant concern. Multimeric enzymes
may be inactivated due to the dissociation of the enzyme subunits.[17,18] For multimeric
enzymes, the subunit–subunit interactions maintain the quaternary structures. However,
the interactions are reduced under certain experimental conditions, leading to
decreasing the enzymatic activity.[19] The moderate-to-low stability of multimeric
enzymes hinders their applications at industrial scale. The multimeric enzymes RTA
and DAAO also face the same concern of stability.
Activity assay has shown that RTA&DAAO and RTA#DAAO can catalyze
transamination and deamination, indicating that the enzymatic activities of RTA and
DAAO have been retained by RTA&DAAO and RTA#DAAO. It is implied that after
the in vivo subunit splicing and in vitro non-covalent association, the original
quaternary structures of RTA and DAAO have been preserved. Circular dichroism (CD)
spectra were measured to monitor the change of the secondary structures of the enzymes
after the in vivo subunit splicing and the in vitro subunit association. Figure 5 shows
7
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ChemBioChem
10.1002/cbic.201800646
the CD spectra for RTA&DAAO, RTA#DAAO, and the mixed enzymes RTA+DAAO
control). The intensity difference between three CD spectra are very small. Figure 5
(
demonstrates that the in vivo subunit splicing and the in vitro non-covalent association
had little effect on the secondary structures of the enzymes.
Fluorescence spectra were measured for RTA&DAAO, RTA#DAAO, and
RTA+DAAO using urea as denaturing agent. To investigate the conformational change
upon interfering by urea, the intrinsic fluorescence of the two-enzyme systems was
quenched by acrylamide. The fluorescence spectra with excitation of 295 nm are shown
Figure 6a, 6b, and 6c. The concentration-dependence quenching was evaluated by the
Stern-Volmer equation F
0
/F=1+ KSV[C],[20] where F
and F are the fluorescence
0
intensities in the absence and presence of acrylamide, respectively. [C] is the
acrylamide concentration. The Stern-Volmer quenching constant KSV was compared to
investigate the conformational change.[20] A larger KSV value is obtained when the
protein has a larger conformational change. Figure 6d shows the Stern-Volmer plots for
the two-enzyme systems. The KSV value for RTA&DAAO at the urea concentration (4
M) is 7.61, which is smaller than 9.12 for RTA#DAAO and 10.29 for RTA+DAAO.
Both RTA&DAAO and RTA#DAAO are more stable than the mixed enzymes
RTA+DAAO (control). In comparison to RTA&DAAO, RTA#DAAO is more
inclined to expose the tryptophan residues when interfered by urea. The association
product RTA#DAAO has a lower stability than the splicing product RTA&DAAO upon
the urea denaturation. RTA&DAAO resulted from the covalent bonding of the RTA and
DAAO subunits, in contrast to RTA#DAAO obtained from the coiled-coil association.
The BN-PAGE analysis based on Figures 3c and 4b show that, the number of the RTAS-
S
S
S
DAAO subunit in RTA&DAAO is 23.1% higher than that of the RTA =DAAO
subunit in RTA#DAAO (per gram of enzymes). This led to a lower mobility of the
8
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ChemBioChem
10.1002/cbic.201800646
subunits in RTA&DAAO, which may become more rigid than RTA#DAAO.
Enzymatic activity
The enzymatic activity for catalyzing the D-alanine deamination reaction by DAAO
and RTA&DAAO was investigated, in order to investigate the effect of the hetrosubunit
ligation on the catalysis efficiency. In the deamination reaction, D-alanine is converted
to pyruvate. 2,4-Dinitrophenylhydrazine (DNPH) can react with pyruvate to produce a
coloured product.[21] It was added to the reaction systems for the qualitative comparison
of enzymatic activity. Both the solutions of DAAO and RTA&DAAO exhibited a
colour in yellow (Figure S6a), there is no significant difference in the colour intensity
between the DAAO and RTA&DAAO solutions. Figure S6 indicated that
RTA&DAAO exhibited almost the same capability with DAAO for catalyzing the
deamination of D-alanine. Furthermore, a quantitative comparison was performed by
studying the enzyme kinetics. The kinetics of RTA&DAAO was compared with that of
DAAO. Figure S6b shows the plots of the initial rate of reaction versus the
concentration of D-alanine. The kinetics parameters were obtained by regressing the
data using the Michaelis-Menten kinetics equation. RTA&DAAO exhibited a Km value
1
5.88 mM, which is comparable to that 15.37 mM by DAAO, indicating that the effect
of the ligation of hetrosubunits on the affinity towards the substrate is very small.
-1
RTA&DAAO exhibited a Kcat value of 1115.5 min , in comparison to the Kcat value
-1
of 1155.8 min by DAAO, indicating that RTA&DAAO exhibited a turnover number
being comparable with that by DAAO. The Kcat/Km ratio is a measure of catalysis
efficiency.[22] The Kcat/Km ratio of RTA&DAAO is 70.3 mM min , which is 93.4%
-1
-1
-1
-1
of that of DAAO 75.2 mM min . It is indicated that the ligation of hetrosubunits has
a very small effect on the catalysis efficiency of DAAO.
In R-ω-transaminase catalyzing reactions, amine conversion can be carried out by
9
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ChemBioChem
10.1002/cbic.201800646
using pyruvate as an amine acceptor, and D-alanine is the resulting co-product. It is
better to maintain pyruvate at a low concentration in order to facilitate subsequent
[
8]
product purification. On the other hand, in situ removal of D-alanine eliminates the
by-product inhibitory effect. In the enzymatic activity assay, a small amount of pyruvate
(
0.5 mM) was used, which is tenth of that of (R)-amines (5 mM). The transamination
of amines was catalyzed under RTA&DAAO, RTA#DAAO, and RTA+DAAO. Figure
a shows the profile of furfurylamine conversion versus reaction time. The control
reaction using RTA+DAAO achieved a conversion of 11.1% after 6 h reaction, and
8.5% of furfurylamine was converted under the catalysis of RTA#DAAO. For these
8
1
two-enzyme systems, after 5 h reactions, the conversions reached a plateau. In contrast,
RTA&DAAO achieved a furfurylamine conversion of 56.3% after 6 h reaction, much
higher than the conversions achieved by RTA#DAAO and RTA+DAAO. Figure 7a
shows that RTA&DAAO exhibited much higher catalysis efficiency than RTA#DAAO
and RTA+DAAO for the conversion of furfurylamine. Figure 7b shows the result of
the conversion of (R)-2-aminooctane. The conversions were 77.9%, 58.4, and 32.3%
after 6 h reactions for RTA&DAAO, RTA#DAAO, and RTA+DAAO, respectively,
indicating the advantage of RTA&DAAO over RTA#DAAO and RTA+DAAO. For
the conversions of phenethylamine (Figure 7c), RTA&DAAO exhibited a higher
catalysis efficiency than RTA#DAAO, and much higher efficiency than RTA+DAAO
(control).
The three two-enzyme systems were used for the resolution of -
methylbenzylamine. The conversion and enantiomeric excess are shown in Figure S7,
closed symbols showing enantiomeric excess and open symbols presenting conversion.
The results of conversion and enantiomeric excess show that the splicing product
RTA&DAAO needed 80 min to reach 50% conversion yielding the S-product (99%
e.e), in contrast to 120 min required by RTA#DAAO and 180 min needed by
RTA+DAAO. The results demonstrated the splicing product RTA&DAAO exhibited a
10
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ChemBioChem
10.1002/cbic.201800646
higher catalytic efficiency than RTA#DAAO and RTA+DAAO for the kinetic
resolution of high concentration of racemic amine.
Discussion
Under the catalysis of the two-enzyme systems, the by-product D-alanine can be
deaminated back to pyruvate under the catalysis of DAAO, achieving the recycling of
pyruvate. In a separate experiment, we performed the RTA catalysis by using an excess
amount of pyruvate (6 mM) in comparison to amines (5 mM), the catalysis efficiency
was lower than that under the catalysis of RTA together with DAAO with a catalytic
amount of pyruvate (0.5 mM). It is ascribed to that the co-substrate pyruvate has an
inhibitory effect on the transamination reaction.[8,10] Hence the recycling of pyruvate is
necessary for the transamination reaction. For the mixed two enzymes RTA+DAAO,
there are no specific interactions between the two enzymes. In the period of reaction
time, the enzyme molecules moved randomly in the reaction solution, and the pairing
of RTAand DAAO might be occasionally occurred due to the enzyme motion. However,
such kind of enzyme pairing is instantaneous and cannot be lasted for a long time due
to the lack of specific interaction. As a result, the diffusion and transferring of D-alanine
and pyruvate between RTA and DAAO cannot be directional, and the pyruvateD-
alaninepyruvate cycle cannot be formed for the mixed two enzymes RTA+DAAO.
In contrast, RTA&DAAO was obtained via in vivo splicing of hetrosubunits (a covalent
approach to linking the hertrosubunits), RTA#DAAO was a result of association of
hetrosubunits (a non-covalent approach to associate the hertrosubunits). Both
RTA&DAAO and RTA#DAAO achieved the pairing of the RTA and DAAO subunits,
as illustrated in Figures 1 and 2. The distance travelled by D-alanine and pyruvate
between RTA to DAAO is short due to the subunit pairing, facilitating the transfer of
D-alanine and pyruvate. The diffusion and transferring of D-alanine and pyruvate are
11
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ChemBioChem
10.1002/cbic.201800646
S
S
directional. In RTA&DAAO, the paired subunits RTA -DAAO were formed by the
direct ligation of two terminal linkers as illustrated in Figure 1 and Figure 8a. While in
S
S
RTA#DAAO, the paired subunits RTA =DAAO were formed by the association
interaction of two terminal peptides Z and Z as shown in Figure 2 and Figure 8b.
Using the software Discovery Studio 4.5, the length of the linker is 3.3 nm, and the
lengths of the peptides Z and Z are 7.9 and 7.2 nm, respectively. The largest distance
between the paired subunits of RTA and DAAO in RTA&DAAO is 6.6 nm (3.3+3.3
R
E
R
E
S
S
S
S
nm, Figure 8a). The smallest distance between the paired subunits of RTA and DAAO
in RTA#DAAO is 14.5 nm {6.6 nm + 7.9 nm (the smallest length of associated peptides,
and Z being completely overlapped)} (Figure 8b). In RTA&DAAO, the diffusion
and transferring of D-alanine and pyruvate are highly directional due to the shorter
Z
R
E
S
S
S
S
distance between the paired subunits of RTA and DAAO (RTA -DAAO ) as
indicated in Figure 8a. Thus, forming the pyruvateD-alaninepyruvate cycles is
S
S
highly efficient. The distance between the paired subunits of RTA and DAAO
S
S
(
RTA =DAAO ) in RTA#DAAO is larger than that in RTA&DAAO, and pyruvate and
S
S
D-alanine are transferred between RTA and DAAO by travelling a larger distance. It
can be speculated that the transferring efficiency of pyruvate and D-alanine by
RTA&DAAO is larger than that by RTA#DAAO. In addition, the BN-PAGE analysis
(Figures 2 and 3) for RTA&DAAO and RTA#DAAO has provided the information of
linking modes corresponding to the bands, the fraction of each band, and apparent
molecular weight. Based on these information, the number of the key subunit RTAS-
S
DAAO (paired subunits via a peptide bond) is 23.1% higher than that of the key
S
S
subunit RTA =DAAO (paired subunits via coiled-coil association). This implies that
RTA&DAAO can generate more pyruvateD-alaninepyruvate cycles than
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10.1002/cbic.201800646
RTA#DAAO. Highly efficiently forming more pyruvateD-alaninepyruvate cycles
contributes to the efficient utilization of pyruvate, this has a significant effect on the
transamination reaction.
Conclusions
For enzymatic catalysis of transamination of amines, efficient utilization of the amino
acceptor pyruvate can significantly contribute to the improvement of the transamination
reaction. Three two-enzyme systems were studied to investigate how pyruvate can be
used efficiently, including RTA&DAAO (via in vivo splicing of hetrosubunits, a
covalent approach to linking hertrosubunits), RTA#DAAO (via association of
hetrosubunits, a non-covalent approach to linking hertrosubunits), and RTA+DAAO
(mixing two enzymes). For the system RTA+DAAO, there is no specific interaction
between RTA and DAAO. The diffusion and transferring of D-alanine and pyruvate
cannot be directional, and the pyruvateD-alaninepyruvate cycle cannot be formed
S
for RTA+DAAO. In RTA&DAAO, the subunits of RTA were specifically ligated to
S
the subunits of DAAO in a one-by-one fashion, thus the heterosubunits are close to
each other at a molecular distance. The diffusion and transferring of D-alanine and
pyruvate between RTA and DAAO can be highly directional, and the pyruvateD-
alaninepyruvate cycle can be formed highly efficiently. In RTA#DAAO, the subunits
S
S
of RTA and DAAO are paired through specific association. The transferring of
pyruvate and D-alanine is directional. The distance between the paired subunits of RTAS
S
S
S
and DAAO (RTA =DAAO ) in RTA#DAAO is larger than that in RTA&DAAO, and
S
S
pyruvate and D-alanine are transferred between RTA and DAAO by travelling a larger
distance. It can be speculated that the transferring efficiency of pyruvate and D-alanine
by RTA&DAAO is larger than that by RTA#DAAO. The BN-PAGE analysis has
S
S
demonstrated that the number of the key subunit RTA -DAAO (paired subunits via a
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ChemBioChem
10.1002/cbic.201800646
peptide bond) in RTA&DAAO is 23.1% higher than that of the key subunit
RTA =DAAOS (paired subunits via coiled-coil association) in RTA#DAAO.
S
RTA&DAAO can generate more pyruvateD-alaninepyruvate cycles than
RTA#DAAO. Highly efficiently forming more pyruvateD-alaninepyruvate cycles
contributes to the efficient utilization of pyruvate, this has a significant promoting effect
on the transamination reaction. For the transamination of amines, RTA&DAAO
exhibited a higher catalysis efficiency than RTA#DAAO, and a much higher catalysis
efficiency than the mixed enzymes RTA+DAAO.
Experimental section
Materials
DNA ligase, DNA polymerase, and restriction enzymes were purchased from New
England Biolabs and Fermentas. Oligonucleotide primers were synthesized by BGI
Tech. Escherichia coli strain DH5α was used as a host for DNAmanipulation, and strain
BL21 was used as a host for expressing enzymes. All other reagents were purchased
from Sigma-Aldrich (Shanghai, China) and used without purification.
Construction of plasmids
Plasmid for the in vivo subunit splicing. Standard molecular cloning techniques were
used for the construction of plasmids. The gene of D-amino acid oxidase (DAAO) was
amplified by PCR using the paired primers DAAO-fp and DAAO-rp (supplementary
Table S1), and the genomic DNA of Trigonopsis variabilis was used as template. A
DNA extraction kit (Omega Bio-tek) was used to purify the DAAO gene. The purified
DAAO gene was digested with both NdeI and KpnI restriction endonucleases, and then
was ligated into the plasmid pETDuet-1 that had been cleaved with NdeI and KpnI
restriction endonucleases. The expression plasmid DAAO-pETDuet in Escherichia coli
C
was constructed, comprising an N-terminal hexahistidine tag. The Int -link gene was
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ChemBioChem
10.1002/cbic.201800646
C
C
amplified by PCR using the Int -fp and Int -rp primers (supplementary Table S3). The
C
purified Int -link gene was digested with KpnI and XhoI restriction endonucleases, then
was ligated into the vector DAAO-pETDuet that had been cleaved with KpnI and XhoI
restriction endonucleases. The constructed expression plasmid was designated as
C
DAAO-limk-Int -pETDuet.
R-ω-transaminase (RTA) gene was synthesized and cloned in pUC57 plasmid by
Genewiz (Suzhou, China). The RTA gene was amplified by PCR using primers RTA-fp
and RTA-rp (supplementary Table S3). The purified RTA gene was digested with both
BamHI and HindIII restriction endonucleases, and then was ligated into the plasmid
N
DAAO-limk-Int -pETDuet, which had been cleaved with BamHI and HindIII
N
restriction endonucleases. Thus, the vector DAAO-limk-Int /RTA-pETDuet in
C
Escherichia coli was constructed. The gene of Int -link was amplified by PCR using
C
C
C
the Int -fp and Int -rp primers (supplementary Table S3). The purified Int -link gene
was digested with both BamHI and NcoI restriction endonucleases, and then was ligated
N
into the plasmid DAAO-limk-Int /RTA-pETDuet, which had been cleaved with BamHI
N
C
and NcoI restriction endonucleases. The expression plasmid DAAO-link-Int /Int -link-
RTA-pETDuet was finally constructed as illustrated in supplementary Figure S1, which
was confirmed by sequence analysis. For expressing RTA, the vector RTA-pET28a was
constructed comprising a C-terminal hexahistidine tag.
Plasmids for the fusions of Z
R
to RTA and Z to DAAO. The gene of DAAO was
E
amplified by PCR using the paired primers DAAO-ffp and DAAO-rfp (supplementary
Table S4). The purified DAAO gene was digested with both NdeI and XhoI restriction
endonucleases, and then was ligated into the plasmid pET28 that had been cleaved with
NdeI and XhoI restriction endonucleases. The gene of the Link-C
R
-C -Link peptide
E
(
supplementary Table S5) was synthesized by BGI (Shenzhen, China). The gene of
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ChemBioChem
10.1002/cbic.201800646
Link-C
then was ligated into the plasmid DAAO-pET28 that had been cleaved with NcoI and
NheI restriction endonucleases. Thus the plasmid C -DAAO-pET28 was constructed
R
-C -Link was digested with both NcoI and NheI restriction endonucleases, and
E
E
(supplementary Figure S4a).
The RTA gene was amplified by PCR using primers RTA-ffp and RTA-rrp
(supplementary Table S4). The purified RTA gene was digested with both NheI and
BamHI restriction endonucleases, and then was ligated into the plasmid pET28, which
had been cleaved with NheI and BamHI restriction endonucleases. The gene of Link-
C
R
-C
was ligated into the plasmid RTA-pET28 that had been cleaved with SacI and XhoI
restriction endonucleases. Thus the plasmid C -RTA-pET28 was constructed
supplementary Figure S4b).
Enzyme expression and in vivo subunit splicing
E
-Link was digested with both SacI and XhoI restriction endonucleases, and then
R
(
Escherichia coli BL21 cells harboring various plasmids grew in Luria-Bertani medium
with 50 µg/ml Amp at 37 °C. When reaching an OD600 of 0.5, the cells were induced
with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and an additional 8 h was
C
required for expressing enzymes at 25°C for coexpressing the precursors Int -link-RTA
N
and DAAO-link-Int and the in vivo splicing. The Escherichia coli cells were harvested
by centrifuging at 6000 g at 4°C for 30 min. The cells were then lysed under
ultrasonication on ice, and cell debris was removed by centrifugation of the lysate at
8
,000 g at 4 °C for 30 min. The supernatant was transferred to a fresh tube. The enzymes
were purified using Ni Sepharose beads (GE Life Sciences) in a chromatography
column (GE Life Sciences) with 500 mM imidazole as the elution liquid. The imidazole
was then removed from the purified enzymes by dialysis into a buffer (500 mM NaCl,
5
0 mM Tris-Cl pH 7.5, 1 mM EDTA). The purified enzyme comprising the splicing
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10.1002/cbic.201800646
product RTA&DAAO was further fractionated by ultrafiltration (GE Healthcare, 50
KDa cut off). The fusion enzymes Z -RTAand Z -DAAO were expressed similarly and
R
E
purified using Ni Sepharose beads.
Blue native PAGE
Blue native PAGE (BN-PAGE) was performed at room temperature using 3.5%
stacking gel and 4–12% separating gel with the gel dimensions 0.15×10×10 cm.[23] The
gel compositions and the cathode and anode buffers were prepared according to the
paper.[24] Prior to use, the buffers were stored at 4 °C. The marker protein standard
(
Invitrogen) consisted of seven protein components with molecular weights from
6~1236 kDa. 2 μg enzyme sample was added to each lane with an enzyme
concentration of 0.5 mg/mL. The BN-PAGE electrophoresis was initially performed at
00 V. After enzyme reaching the separating gel, the voltage was changed to 160 V and
6
1
run until the enzyme front reached the end of the gel. Once the blue running front was
moved about one-third to half of the separating gel, the buffer was changed to a low
concentration.
The obtained gels were scanned and high resolution images (600 dpi) were obtained.
The gel images were processed using Adobe Photoshop CS6 (version 13) to obtain the
best contrast for densitometry analysis. The Gel-Pro Analyzer software program was
used to estimate optical densitometry for determining relative abundances of the
RTA&DAAO fractions. By comparing the electrophoretic mobility of the enzyme
fractions with that of Marker proteins, the apparent molecular weights of the
RTA&DAAO fractions were determined. The relative mobility was determined by
measuring the distance from the top of the gel to the middle of the dye front, and
dividing the second measurement by the first. The relative mobility versus molecular
weight for the Marker proteins is shown in supplementary Figure S3. The curve was
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ChemBioChem
10.1002/cbic.201800646
used as a standard curve for determination of the apparent molecular weights of the
RTA&DAAO fractions.
Circular dichroism (CD) spectra
The change of secondary structures of the enzymes was monitored by measuring CD
spectra on a JASCO J-810 CD instrument. The scanning was performed at 30 °C with
a scan rate of 100 nm/min with a bandwidth of 1.0 nm. Cell length was 1 cm, and the
enzyme concentration was 0.2 mg/mL. All the samples were prepared by dissolution of
the enzymes in PBS buffer. The CD spectrum of the PBS buffer was measured and used
as control. Averaged spectra were obtained by repeating five times scan.
Fluorescence measurements
Two buffers were first prepared, buffer A (pH 7.5) consisting of 50 mM potassium
phosphate and 2 mM EDTA, and buffer B (pH 7.5) comprising 50 mM potassium
phosphate, 2 mM EDTA, and 4 M urea. The enzyme solutions were diluted into buffer
0
A and buffer B to prepare samples, incubated at 4 C overnight, with a final enzyme
concentration of 2.0 mol/L each. Buffer A was used to prepare acrylamide solutions
with different concentrations. Fluorescence spectra were measured on a
spectrophotometer (F-7000), a 1 cm pathlength cuvette was used. Emission spectra of
the tryptophan were recorded for wavelength ranging from 300 to 400 nm, using an
excitation wavelength of 295 nm. The slits for excitation and emission were set at 5 nm,
the scan speed was 1200 nm/min. For all the samples, the background fluorescence was
subscribed to correct the fluorescence spectra. A baseline control was realized for each
experiment. The experiments for fluorescent quenching were performed by addition of
the acrylamide solutions. A aliquot of acrylamide solution was added to the samples (2
mL), incubation of the solutions was carried out for 6 min prior to recording the
emission spectra.
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10.1002/cbic.201800646
Enzymatic activity
For transformation of the amines, the enzymatic catalysis was performed at 30 °C. The
enzyme concentrations were: 0.2 mg/mL RTA&DAAO; 0.089 mg/mL RTA-Z + 0.111
mg/mL DAAO-Z ; 0.088 mg/ml RTA+0.112 mg/ml DAAO. For the catalysis by
R
E
RTA&DAAO and RTA+DAAO, the substrate solutions were prepared by dissolving
amine (5 mM), pyridoxal-5’- phosphate (1 mM), and sodium pyruvate (0.5 mM) in
Tris-HCl (100 mM, pH 8.5). For the catalysis by RTA#DAAO, the substrate solutions
were prepared by dissolving amine (5 mM), pyridoxal-5’- phosphate (1 mM), and
sodium pyruvate (0.5 mM) in KPOi (100 mM, pH 7.5). Enzyme concentrations were
determined by the bicinchoninic acid assay. Amines were quantified by HPLC using a
C18 column (Diamonsil). The mobile phase was acetonitrile/acidic acid/water
(60:0.1:39.9 by vol) at 0.8/1.0 ml/min. The injection was 20 ul.
Acknowledgments
This work was supported by The National Science Foundation of China (21576018,
2
1776011).
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Figures
Figure 1. Schematic presentation of subunit splicing for the ligation of the RTA and
DAAO subunits with peptide bond.
Through the intein-mediated subunit splicing, the N-terminus of the RTA subunit is specifically ligated
to the C-terminus of the DAAO subunit with native peptides. In the splicing product (RTA&DAAO), the
S
S
RTA DAAO subunit is the key subunit. The DAAO and RTA subunits, that are not involved in the
S
S
subunit splicing reaction, are associated with the RTA DAAO subunit.
Figure 2. Schematic presentation for establishing two-enzyme system RTA#DAAO
through coiled-coil association of Z
In the association product (RTA#DAAO), the RTA =DAAO subunit is the key subunit.
E
and Z in vitro.
R
S
S
ZR: arginine-rich leucine zipper; ZE: glutamic acid-rich leucine zipper
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ChemBioChem
10.1002/cbic.201800646
Figure 3. (a) Analysis of the splicing product RTA&DAAO by SDS-PAGE.
b) Analysis of the splicing product RTA&DAAO by BN-PAGE.
Lane M: marker.
(
(c) Schematic diagram representing the RTA&DAAO fractions resulted from the
23
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ChemBioChem
10.1002/cbic.201800646
ligation modes of
RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO
RTA (band I); RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA~DAAO~RTA (band II); DAAO~
~
RTA~DAAO~RTA~ DAAO~RTA~DAAO (band III); RTA~DAAO~RTA~DAAO~RTA~DAAO~
RTA (band IV); RTA~DAAO~RTA~DAAO~RTA (band V); DAAO~RTA~DAAO (band VI).
Figure 4. (a) Analysis of the association product RTA#DAAO by BN-PAGE.
(
b) Schematic diagram representing the RTA#DAAO fractions resulted from the
association modes of
RTA≈DAAO≈RTA≈DAAO≈RTA≈DAAO≈RTA (band I);
RTA≈DAAO≈RTA≈DAAO≈RTA (band II);
DAAO≈RTA≈DAAO (band III).
Figure 5. CD spectra for the splicing product RTA&DAAO, association product
RTA#DAAO, and mixed enzymes RTA+DAAO.
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ChemBioChem
10.1002/cbic.201800646
Figure 6. (a, b, c) Change of fluorescence intensity of the two-enzyme systems with
acrylamide concentration (M). (d) Stern-Volmer plots for the quenching of protein
fluorescence with acrylamide. F and F are the fluorescence intensities in absence and presence
0
of acrylamide, respectively.
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ChemBioChem
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Figure 7. Conversion of (R)-amines under the catalysis of two-enzyme systems
Substrates: (a) Furfurylamine; (b) (R)-1-Aminoindan; (c) (R)-Phenethylamine
For catalyzing the transamination reactions, equal molar ratio of RTA to DAAO was adopted for the
two-enzyme system RTA&DAAO, RTA#DAAO, and RTA+DAAO (see experimental section).
The concentrations of amines were 5 mM, and the concentration of pyruvate was 0.5 mM.
For RTA&DAAO, the mixture of bands I~VI was used. For RTA#DAAO, the mixture of the mixture
of band I~III was used.
Figure 8. Schematic presentation for the coordination of RTA and DAAO by the two-
enzyme systems. (a) RTA&DAAO; (b) RTA#DAAO
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Table of contents text
The N-terminus of the subunit of R-ɷ-transaminase is specifically ligated to the C-
terminus of the subunit of D-amino acid oxidase with native peptides. The hetrosubunits
are spatially arranged in a one-by-one fashion at a molecular distance. The
pyruvateD-alaninepyruvate cycles are efficiently formed, promoting the
transamination reaction forward.
27
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