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Applied and Environmental Microbiology, May 2000, p. 2133-2138, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Construction and Evaluation of a Novel
Bifunctional N-Carbamylase-D-Hydantoinase
Fusion Enzyme
Geun-Joong
Kim,
Dong-Eun
Lee, and
Hak-Sung
Kim*
Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, 373-1, Kusung-dong,
Yusung-gu, Taejon 305-701, Korea
Received 1 November 1999/Accepted 20 February 2000
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ABSTRACT |
A fully enzymatic process employing two sequential enzymes,
D-hydantoinase and N-carbamylase, is a typical
case requiring combined enzyme activity for the production of
D-amino acids. To test the possibility of generating a
bifunctional fusion enzyme, we constructed a fusion protein via
end-to-end fusion of a whole gene that encodes an intact protein at the
N terminus of the D-hydantoinase. Firstly, maltose-binding
protein (MBP) gene of E. coli was fused with
D-hydantoinase gene from Bacillus
stearothermophilus SD1, and the properties of the resulting
fusion protein (MBP-HYD) were compared with those of native
D-hydantoinase. Gel filtration and kinetic analyses clearly
demonstrated that the typical characteristics of
D-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of
the whole length of N-carbamylase (304 amino acids [aa])
from Agrobacterim radiobacter NRRL B11291 and
D-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was
functionally expressed with an expected molecular mass of 86 kDa and
efficiently converted exogenous hydantoin derivatives to the
D-amino acids. A related D-hydantoinase (HYD1)
gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The
resulting enzyme (CAB-HYD1) was bifunctional as expected and showed
better performance than the CAB-HYD fusion enzyme. The conversion of
hydantoin derivatives to corresponding amino acids by the fusion
enzymes was much higher than that by the separately expressed enzymes,
and comparable to that by the coexpressed enzymes. Thus, the fusion
enzyme might be useful as a potential biocatalyst for the production of
nonnatural amino acids.
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INTRODUCTION |
In the field of molecular biology
and biotechnology, enzymes possessing two or more combined activities,
along with appropriate stability, have found wide application
(25). Although the natural diversity of the enzymes provides
some candidates that have evolved to possess bifunctional activity,
most fusion enzymes have resulted from the in vitro fusion of
individual enzymes based on evolutionary traits and well-defined
structure (1, 25). Artificial fusion enzymes, simply
generated by either end-to-end fusion or by tethering of whole genes
encoding intact functional proteins with a linker, have been reported
to show noticeable performance in a concerted fashion (6,
21).
Nonnatural D-amino acids are widely used in the
pharmaceutical field, with applications such as antimicrobial and
antiviral agents, artificial sweeteners, pesticides, and pyrethroids
(26, 28, 30). Due to the great commercial demand for various
D-amino acids, enzymatic and chemoenzymatic routes have
been developed (29). Of these, a fully enzymatic process
using two sequential enzymes, D-hydantoinase and
N-carbamylase, is a typical case requiring combined enzyme
activity (8). In this process, hydantoin derivative is
hydrolyzed by D-hydantoinase, and the resulting
N-carbamyl-D-amino acid is further converted to
the corresponding D-amino acid by N-carbamylase.
Therefore, functional fusion of two enzymes was expected to have
several advantages over individual enzymes with respect to reaction
kinetics and enzyme production, as well as novel properties and
reactivity. As practical cases of the multistep sequential reaction,
the performance of the hybrid or fusion enzymes sometimes was better
than that achieved by successive action of individual enzymes,
expanding the potential use of natural enzymes (6, 21, 25).
Previously, we cloned and expressed two hydantoinase genes from
Bacillus stearothermophilus SD1 (17) and
Bacillus thermocatenulatus GH2 (15). We also
identified some conserved domains possessing essential amino acid
residues by comparative analyses of functionally related enzymes
(16). These results provided some evidence that supports the
presence of a cyclic amidohydrolase family including hydantoinase,
dihydropyrimidinase, allantoinase, and dihydroorotase. Further study of
the deletion mutants derived from two D-hydantoinases suggested that the N terminus of D-hydantoinase is not
essential for maintaining the enzyme structure and is dispensable for
enzyme activity (15, 17). From these observations, we made a
hypothesis that the hybrid enzymes might be generated via the linear
fusion of a protein at the N terminus of D-hydantoinase,
resulting in a bifunctional fusion enzyme.
Here we report the generation of fusion enzymes by end-to-end fusion of
a whole gene that encodes an intact protein to the N terminus of the
D-hydantoinase from B. stearothermophilus SD1 or
to that of B. thermocatenulatus GH2. Based on the distinct fusion ability of D-hydantoinase observed in
maltose-binding protein (MBP) fusion, we constructed a bifunctional
fusion enzyme composed of N-carbamylase and
D-hydantoinase. The performance of the resulting fusion
enzymes was evaluated and compared with the performances of the
coexpressed and separately expressed enzymes. Details are reported herein.
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MATERIALS AND METHODS |
Construction of fusion enzymes.
Restriction enzymes, the
pMAL-c2X vector, and amylose resin were obtained from New England
Biolabs. Primers SDN (5'-TAGAATTCATGACAAAAATTATAAAAAATC-3') and SDC (5'-TACTGCAGTTAAATGGTTAATTCCTCGCTC-3') and
primers GHN (5'-TAGAATTCATGACAAAATTGATAAAAAATG-3') and GHC
(5'-TACTGCAGTTAGGACATTTTCACCACATCT-3'), spanning the
genes encoding the D-hydantoinase
from B. stearothermophilus SD1 (17) and B. thermocatenulatus GH2 (15), respectively, were used.
Restriction sites EcoRI and PstI were introduced
into the N- and C-terminal primers, respectively. The amplified DNA fragments (1.4 kb) encoding the D-hydantoinases were cloned
into the EcoRI/PstI site of pMAL-c2X, and the
resulting plasmids were transformed into Escherichia coli JM109.
For the construction of
N-carbamylase-D-hydantoinase fusion enzymes,
the Agrobacterium N-carbamylase gene (7) was
amplified from chromosomal DNA with two primers, CAN
(5'-CAAAGGTCCATGGCACGTCAGATGATA-3') and CAC
(5'-AAGGGATCCTTATCAGAATTCCGCGATCAG-3'), as N- and C-terminal primers, respectively. The resulting fragment was inserted into the
NcoI and BamHI sites of pTrc99A, yielding plasmid
pTC. The naturally occurring N-carbamylase gene has a unique
EcoRI restriction site in its C terminus (7),
neighboring the stop codon TAA. The amplified
D-hydantoinase genes from B. stearothermophilus SD1 and B. thermocatenulatus GH2 were inserted to replace
the sequence between the EcoRI and PstI sites in
plasmid pTC, yielding plasmids pTCH and pTCH1, respectively. The
resulting fusion genes (2.3 kb) encoding the fusion enzymes, CAB-HYD
and CAB-HYD1, were confirmed by DNA sequencing. HYD denotes the
D-hydantoinase from B. stearothermophilus SD1,
while HYD1 denotes that from B. thermocatenulatus GH2.
Coexpression of N-carbamylase and
D-hydantoinase in a single host.
For the comparison
with the fusion enzyme, coexpression of N-carbamylase and
D-hydantoinase in a single host was conducted under the
control of Ptrc. The amplified D-hydantoinase gene used in
MBP fusion was cloned into the EcoRI and PstI
sites in plasmid pTrc 99A to generate pTH. A DNA fragment containing
the inserted gene with Ptrc was digested with NarI and
HindIII, blunt ended, and cloned into the EcoRV
site of pACYC184 (New England Biolabs) to yield plasmid pYH. For the
coexpression, E. coli JM109 was transformed by
electrotransformation in 10% glycerol with two plasmids, pTC and pYH.
Cells were maintained and induced in Luria-Bertani (LB) medium
containing ampicillin (50 µg/ml) and chloramphenicol (25 µg/ml) at
30°C.
Expression and purification of fusion enzymes.
Expression of
the fusion enzymes MBP-HYD and MBP-HYD1 in E. coli JM109 was
achieved through by isopropyl-
-D-thiogalactopyranoside (IPTG) (0.5 mM) induction at 37°C. The fusion enzymes were purified and cleaved with factor Xa according to the general procedure of the
supplier (New England Biolabs). Plasmids pMAL-c2X contains a sequence
coding for the recognition site (Ile-Glu-Gly-Arg) of a specific
protease, factor Xa, allowing the fused protein to be cleaved from MBP.
For comparison, the D-hydantoinases were further purified
from the fusion enzyme by treating with factor Xa, followed by loading
onto amylose resin.
The fusion enzymes CAB-HYD and CAB-HYD1 were expressed in
E. coli JM109 under the control of Ptrc and purified using an
antibody
raised against the purified
D-hydantoinase from
B. stearothermophilus SD1 (
19).
E. coli cells were cultivated in 200 ml of LB broth
containing
ampicillin (25 µg/ml) at 30°C, and IPTG (0.2 mM) was
added for
induction when the optical density at 600 nm (OD
600)
reached about 0.7 to 0.8. After 2 h of cultivation, cells were
collected by centrifugation and then resuspended in 5 ml of 20
mM
Tris-HCl (pH 7.8) buffer containing 0.1 mM manganese chloride,
0.1%
phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton X-100,
and 1 mM
dithiothreitol (DTT). Both fusion proteins were purified
using the
following protocol. Suspended cells were freeze-thawed
twice and
disrupted by sonication, and the cell lysate was centrifuged
at 27,000 ×
g for 1 h. The supernatant was incubated overnight
with immunoglobulin G (IgG)-immobilized Sepharose 4B (
19)
under
a nitrogen gas atmosphere. After a wash with 20 mM Tris-HCl (pH
7.8) containing 0.25 mM NaCl, immunoabsorbed proteins were eluted
from
the column with 50 mM carbonate buffer containing 2 M NaCl.
Active
enzyme fractions were dialyzed against the buffer (20 mM
Tris-HCl [pH
7.8]) and used for further
analyses.
Oligomeric structure analysis.
The oligomeric structures of
enzymes were determined in a gel filtration column (Superose-12
HR10/30) mounted onto a fast protein liquid chromatography system
(Pharmacia). The flow rate of the mobile phase containing 20 mM
Tris-HCl and 150 mM NaCl was 0.3 ml/min. The column was calibrated
using the native protein markers (Pharmacia), and a molecular mass
standard curve was established using the semilog method based on data
obtained from the elution profile of protein markers (Pharmacia).
Enzyme assay and conversion test.
The activities of
N-carbamylase and D-hydantoinase were determined
at 45°C for 30 min with constant shaking after addition of whole
cells or purified enzymes. In the case of D-hydantoinase activity, either hydantoin or hydroxyphenylhydantoin (HPH) was used at
a final concentration of 15 mM as a substrate in 100 mM Tris-HCl (pH
8.0). For the N-carbamylase activity, 15 mM of
N-carbamyl-hydroxyphenylglycine (NCHPG) in 100 mM potassium
phosphate buffer (pH 7.0) containing 1 mM DTT was used as a substrate.
The conversion experiments were performed in a total volume of 10 ml
containing 15 mM or 20 mM hydantoin derivatives, 100
mM phosphate
buffer (pH 7.2), and 1 mM DTT under a nitrogen atmosphere.
Induced
whole cells (95 mg) were harvested and added into the
reaction vial.
The reaction products were analyzed using high-performance
liquid
chromatography (HPLC) (
14). The expression levels of
the
recombinant enzymes were analyzed by sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) followed by
scanning of gels with
a densitometer (Bio-Rad). Protein concentration
was measured by
using a protein assay solution (Bio-Rad). Kinetic
constants of
the enzyme were also determined as described previously
(
14).
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RESULTS |
Expression and purification of MBP-HYD fusion protein.
As a
preliminary experiment, a 1.4-kb SacI fragment including the
whole open reading frame (ORF) encoding D-hydantoinase from B. stearothermophilus SD1 was inserted into the
SacI site of pGEM-7Zf(+) to create an in-frame fusion of the
D-hydantoinase gene and lacZ. The resulting
fusion enzyme was found to be fully functional (data not shown). This
fusion enzyme carries a fragment encoding an amino-terminal portion of
-galactosidase linked to the N terminus of the
D-hydantoinase gene product. This result provided the
possibility that fusion of a whole gene encoding intact protein to the
DNA encoding N terminus of D-hydantoinase is feasible. The
above presumption was first tested by linear fusion of MBP to the N
terminus of D-hydantoinase from B. stearothermophilus SD1 as shown in Fig. 1A. Free MBP (43 kDa) was expressed in
induced cells harboring the control plasmid. In contrast, the MBP-HYD
fusion protein of the expected molecular mass (95 kDa) was correctly
expressed. The MBP-HYD fusion protein was purified from the cell
extract and treated with a specific protease factor, Xa, resulting in free MBP (43 kDa) and D-hydantoinase (52 kDa) (Fig. 1A).
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FIG. 1.
Expression and gel filtration analysis of the MBP-HYD
fusion protein. (A) Crude extracts of cells expressing the MBP-HYD
fusion protein and the purified MBP-HYD fusion protein were analyzed on
SDS-10% PAGE. Lane 1, crude extract of cells expressing control MBP;
lane 2, crude extract of cells expressing MBP-HYD; lane 3, the purified
MBP-HYD fusion protein; lane 4, free MBP and D-hydantoinase
released from the fusion protein by factor Xa digestion. (B) A protein
mixture (MBP-HYD, MBP, and HYD) and purified MBP-HYD fusion protein
were analyzed on a Superose-12 gel filtration column. Curve 1, protein
mixture; curve 2, purified MBP-HYD. The native size of each protein was
estimated based on the elution profile of standard protein markers as
follows: blue dextran, 2,000 kDa; ferritin, 440 kDa; catalase, 232 kDa;
aldoase, 158 kDa; Fab fragment, 50 kDa. All experiments were repeated
three times, and the shift in elution time was negligible (<0.2
min).
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Characterization of the MBP-HYD fusion protein.
The oligomeric
state of purified MBP-HYD was investigated by using Superose-12 gel
filtration column chromatography. As shown in curve 2 of Fig. 1B, the
fusion protein eluted as a symmetrical peak corresponding to a
tetrameric structure (330 to 360 kDa), indicating the acquisition of a
distinct oligomeric structure. Free MBP and D-hydantoinase
were eluted at the positions corresponding to monomeric (42 to 45 kDa)
and tetrameric (180 to 210 kDa) structures, respectively (curve 1 of
Fig. 1B). No improper bands resulting from high-molecular-weight
aggregates or other oligomeric structures were detected. When the
fusion protein was treated with the zero-order length cross-linker EDC
(2), a distinct protein band appeared at the tetrameric
position on SDS-PAGE (data not shown). These results imply that the
oligomeric structure and/or folding of the D-hydantoinase
domain is not severely affected even though its N terminus is fused
with another protein.
The activity of the MBP-HYD fusion protein toward hydantoin was about
87 µmol/min/mg, and this is quite similar to that of
native
D-hydantoinase (98 µmol/min/mg). The specific activity
of
fusion protein (13.2 µmol/min/mg) toward HPH was also similar
to that
of the native
D-hydantoinase (11.8 µmol/min/mg) at
55°C.
Free MBP showed no activity for the substrates tested. To
examine
the effect of fused MBP on the affinity to HPH, the apparent
Km was determined at pH 8.0 for both MBP-HYD and
D-hydantoinase,
and similar values of 15.3 ± 1.4 and
12.6 ± 1.2 mM were obtained
in the presence and absence of fused
MBP, respectively. These
results indicated that the
D-hydantoinase was functionally fused
with MBP at its N
terminus.
Construction and identification of the bifunctional CAB-HYD fusion
enzyme.
To make a more valuable bifunctional fusion enzyme, we
constructed an artificial fusion enzyme composed of
N-carbamylase and D-hydantoinase (Fig.
2A). E. coli JM109, harboring
the recombinant plasmid pTCH, was induced with 0.2 mM IPTG at 30°C,
and the crude extract of the cells was analyzed by SDS-PAGE (Fig. 2B).
A protein band corresponding to the CAB-HYD fusion enzyme was observed
between 85 and 88 kDa at different induction times (Fig. 2B, lanes 2 and 3). This is consistent with the expected size of the fusion enzyme (86 kDa) calculated from the fusion gene.
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FIG. 2.
Construction and functional expression of the CAB-HYD
fusion. (A) The organization of the fusion gene encoding the
N-carbamylase and D-hydantoinase. Whole amino
acid residues of both open reading frames were conserved, and the total
length of the fusion protein is therefore 775 residues. The nucleotide
sequence of the junction region is shown in bold. (B) Crude extract of
cells expressing the CAB-HYD fusion enzyme was analyzed on SDS-10%
PAGE. Lane 1, control protein marker obtained from the MBP-HYD fusion
protein (MBP-HYD, 95 kDa; HYD, 52 kDa; MBP, 43 kDa); lanes 2 and 3, crude extract after induction for 1 and 2 h, respectively; lane 4, crude extract of cells harboring pTrc 99A without the fusion gene.
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It has been known that artificial multidomain polypeptides occasionally
show a high aggregation tendency when expressed in
E. coli
possessing a posttranslational folding system (
6).
The
CAB-HYD fusion enzyme constructed in this work was one such
case. When
the expressed enzyme in
E. coli was fractionated to
analyze
the enzyme localization, considerable portions were also
found in the
membrane fraction and cell debris. To estimate the
portion of the
fusion enzymes expressed as an insoluble or a soluble
form, the cell
lysate was solubilized in a buffer containing 1
mM EDTA and 1% Triton
X-100. As a result, the dominant portion
(60 to 65%) of the fusion
enzyme was found to be expressed as
a soluble form, showing both
N-carbamylase and
D-hydantoinase
activities. The
expression level of the CAB-HYD fusion enzyme
in
E. coli was
estimated to be about 6 to 8%.
The antibody raised against free
D-hydantoinase was
observed to recognize the CAB-HYD fusion enzyme and was used for the
purification
of the fusion enzyme. As shown in lane 2 of Fig.
3A, a distinct
protein eluted from the
immunoaffinity column appeared at position
86 kDa. This enzyme fraction
catalyzed the conversion of HPH to
D-hydroxyphenylglycine
(
D-HPG) via NCHPG (Fig.
3B), which clearly
indicates the
bifunctional activity of the fusion enzyme. Aside
from the major band
of the intact fusion enzyme, minor polypeptides
of smaller sizes were
also observed. These polypeptides were removed
by using an ion-exchange
column, and the bifunctional activity
of the fusion enzyme was not
changed. The activities of the CAB-HYD
fusion enzyme toward HPH and
NCHPG were estimated to be 5.1 and
1.3 µmol/min/mg, respectively,
which corresponded to about 89
and 37% of the native enzymes under
defined conditions. From the
gel filtration column chromatography, a
major peak corresponding
to high molecular aggregates was observed,
which implies that
independent folding was not maintained in this
fusion enzyme.
More detailed analyses, such as in vitro refolding and
ultracentrifugation,
would address the exact nature of this fusion
protein.
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FIG. 3.
Affinity purification and bifunctional activity of the
CAB-HYD fusion enzyme. (A) The CAB-HYD fusion enzyme was purified using
an immunoaffinity column. After elution from the immunoaffinity column,
the enzyme was analyzed on SDS-12% PAGE. Lane 1, control protein
obtained from the MBP-HYD fusion protein (95 kDa); lane 2, the purified
CAB-HYD fusion enzyme. (B) HPLC chromatogram of the reaction products
obtained by the action of the purified CAB-HYD fusion enzyme. Peak 1, D-hydroxyphenylglycine; peak 2, N-carbamyl-D-hydroxyphenylglycine; peak 3, the
starting substrate, HPH. An octyldecylsilane column (CLC-ODS) was used
with 10% acetonitrile (pH 3.0) as the mobile phase. The flow rate of
the eluent was 1 ml/min. All compounds shown in the chromatogram were
detected at 214 nm and identified with authentic samples.
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Construction of a bifunctional fusion enzyme using
D-hydantoinase of B. thermocatenulatus
GH2.
The D-hydantoinase (HYD1) from B. thermocatenulatus GH2 studied previously was found to have a
remarkable homology to HYD (15). Only 8% of amino acid
residues were mismatched vis-à-vis each other, which led us to
assume that this enzyme can also be used as a fusion partner for the
construction of a bifunctional fusion enzyme. Moreover, the resulting
fusion enzyme was expected to exhibit a different property from that of
the CAB-HYD fusion enzyme.
MBP-HYD1 was first constructed and purified for analysis using the same
procedure as for MBP-HYD. The fusion enzyme was functionally
active,
exhibiting characteristics similar to those of the native
HYD1.
Favorable activity toward HPH (34.7 U/mg) was obtained with
fusion
enzyme MBP-HYD1, which corresponds to about 85% of the
activity of
native HYD1. MBP-HYD1 retained most of the native
property of HYD1 in
terms of its oligomeric structure (a tetramer)
and apparent
Km for HPH (27.2 ± 1.8 and 25.6 ± 1.2 mM were obtained
in the presence and absence of fused MBP,
respectively).
When HYD1 was fused with
N-carbamylase, the bifunctional
activity of the fusion enzyme (CAB-HYD1) was also clearly detected
when
induced with 0.2 mM IPTG at 30°C for 2 h. The activities
of the
CAB-HYD1 fusion enzyme toward HPH and NCHPG were estimated
to be 7.1 and 2.9 µmol/min/mg, respectively, which are 1.4 and
2.2-fold higher
than those of CAB-HYD. As a striking difference
between CAB-HYD and
CAB-HYD1, expression of CAB-HYD1 was dominant
in the insoluble fraction
(data not
shown).
Both CAB-HYD and CAB-HYD1 fusion enzymes also required a reducing agent
such as DTT for the
N-carbamylase activity (
7,
8). The fusion enzymes lost the
N-carbamylase activity
more
readily than the
D-hydantoinase activity. CAB-HYD
retained its
N-carbamylase activity above 50% for about 6 to 7 h at 45°C, while
D-hydantoinase activity was
maintained at more than 75% of the
initial activity. The
N-carbamylase and
D-hydantoinase activities
of
the CAB-HYD1 fusion enzyme were retained about 78 and 95% of
the
initial activities, respectively, even after 6 h under the
identical conditions. The disparity in enzyme stability seems
to be
linked with the unstable nature of
N-carbamylase and
independent
folding between two domains. It has been reported that
N-carbamylase
is susceptible to inactivation under oxidizing
conditions and
is highly sensitive to hydrogen peroxide (
7,
14).
Conversion of hydantoin derivatives using the fusion enzymes.
The ability of the fusion enzyme to convert the hydantoin derivatives
to final amino acids was tested in small-scale reactions at pH 7.2. To
compare the catalytic activities of the CAB-HYD and CAB-HYD1 fusion
enzymes, we expressed both fusion enzymes under defined conditions (0.2 mM IPTG at 30°C for 2 h) and adjusted the protein level to be
equivalent to that of CAB-HYD observed in lane 3 of Fig. 2B. The
conversion of the various cyclic ureides, including hydantoin
derivatives, by both fusion enzymes is shown in Table
1. Hydantoin derivatives with hydrophobic
side chains, such as HPH and phenylhydantoin, were completely converted
by both fusion enzymes, while an exception was observed in
isopropylhydantoin due to the low affinity of
D-hydantoinase (19). For cyclic ureides such as
hydantoin and dihydrouracil, almost complete conversion to their
corresponding N-carbamyl-amino acids was observed, but the
level of final product was very low. N-Carbamylase was
reported to have a low activity toward achiral carbamyl substrates
(31, 32), and this seems to be the reason for the low
conversion yield.
Comparison of the fusion enzyme with the coexpressed and separately
expressed enzymes.
To further evaluate the performance, the level
of final product produced by the fusion enzymes was compared with those
produced by the coexpressed and separately expressed enzymes. For the
separate expression of two enzymes, E. coli strains
expressing either D-hydantoinase or
N-carbamylase were prepared by inserting the corresponding gene into plasmid pTrc 99A. In the case of coexpression, two
plasmids, pTC and pYH, encoding the N-carbamylase and
D-hydantoinase genes, respectively, were transformed into a
single host as described in Materials and Methods. For clear
comparison, all sets of enzymes were expressed under the control of the
identical promoter (Ptrc) and induced with 0.5 mM IPTG at 30°C for
2 h, and the expression levels and activities of each enzyme in
different expression systems were examined. As shown in Fig.
4, no significant difference in the
expression levels was found for N-carbamylase, but the
expression level of D-hydantoinase in the coexpression
system was about twofold lower than that in the separately expressed
enzyme. The CAB-HYD fusion enzyme showed an expression level similar to
that of the separately expressed D-hydantoinase (see also
Fig. 2), but a twofold-lower level than that of separately expressed
N-carbamylase. These differences in the expression level
were well correlated with the differences in the enzyme activities
observed in each system, as shown in Table
2.
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FIG. 4.
SDS-PAGE analyses of the coexpressed and separately
expressed enzymes. Lane 1, crude extract of E. coli cells
coexpressing D-hydantoinase and N-carbamylase;
lane 2, crude extract of E. coli cells expressing
D-hydantoinase; lane 3, crude extract of E. coli
cells expressing N-carbamylase. Total proteins expressed in
each E. coli strain were analyzed on SDS-10% PAGE.
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Since the expression level was dependent on the expression system as
mentioned above, direct and precise comparisons of the
production rates
between the fused and the coexpressed enzymes
were difficult. Thus, we
carried out conversion experiments under
the condition that the total
activity of either
N-carbamylase
or
D-hydantoinase in the reaction mixture was adjusted to be
equivalent.
As can be seen in Table
3,
when the total activity of
N-carbamylase
was fixed at 0.2 U,
the performance by the fusion enzymes (CAB-HYD
and CAB-HYD1) was better
than that by the coexpressed enzymes.
In this case, the low level of
D-hydantoinase activity in the
coexpressed system seems to
result in a low conversion yield.
As the
D-hydantoinase
activity in the coexpressed system was increased
to an equivalent level
(0.45 U) in the fusion enzyme, the performance
by the coexpressed
enzyme was comparable to or better than that
by the fusion enzymes,
probably due to the high level of
N-carbamylase,
leading to
low accumulation of intermediates. Obviously, the conversion
yield by
the fusion enzymes was much higher than that by the separately
expressed enzymes.
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DISCUSSION |
The experimental results shown in this paper clearly demonstrate
that D-hydantoinase participates in the generation of a
bifunctional fusion enzyme. Fusion of MBP to the N termini of
D-hydantoinases did not abolish the activity of the fusion
enzyme, and the resulting fusion enzyme was functionally expressed in
E. coli. As reported so far, many microbial hydantoinases,
including eucaryotic counterpart dihydropyrimidinases, have been
isolated and purified for characterization (3, 13, 19, 24,
27). In these cases, five or six successive steps were required
for purification to homogeneity. We also purified microbial
D-hydantoinase using the conventional procedures and observed that the oligomeric structure might be altered during purification. For example, D-hydantoinase from B. stearothermophilus SD1 was determined to be a dimer in our
previous work (20), but this enzyme was found to be a
tetramer when purified from the MBP-HYD fusion protein. This result
implies that MBP fusion to the microbial D-hydantoinases
facilitates the purification of hydantoinases, expecting that their
native structures remain intact. To confirm this possibility, we
constructed another fusion enzyme using the D-hydantoinase
from B. thermocatenulatus GH2 (15) and found that
the D-hydantoinase also retains its native properties in a
fusion state. Enzymes belonging to a cyclic amidohydrolase family have
similar ORF sizes, and the apparent organization of primary and
secondary structures is also very similar (4, 9, 11, 16, 18,
22). In this sense, the same strategy can be applied to other
related enzymes to construct a fusion protein.
Unnatural D-amino acids are widely used in the synthesis of
semisynthetic antibiotics, peptide hormones, and pesticides. These amino acids currently are produced via two sequential reactions mediated by D-hydantoinase and N-carbamylase
(29, 30). For process development, each enzyme has been
separately expressed in different host cells (4, 12, 17, 18)
or in a single host (8). In this process, the different
expression levels of the two each enzymes, the formation of inclusion
bodies, and the transport of restricted substrates through the cell
membrane remained to be solved. In an effort to produce
D-amino acids in a concerted fashion, we constructed the
bifunctional fusion enzyme composed of N-carbamylase and
D-hydantoinase by an end-to-end fusion method. The
resulting fusion enzymes expressed in a single host showed better
performance than separately expressed enzymes. It seems that the
intermediate produced by the first enzyme is readily available to the
second-step enzyme, leading to a faster reaction rate. From the
comparison with the coexpressed enzyme, the performance by the fusion
enzyme was observed to be comparable, even though precise comparison
needs further kinetic analysis. Thus, both fusion enzymes (CAB-HYD and
CAB-HYD1) may be used as biocatalysts for the production of
D-hydroxyphenylglycine and D-phenylglycine,
D-amino acids which are in great demand. However, the low
stability of the fused N-carbamylase seems to limit the use
of the fusion enzyme in a sequential reaction. As an alternative strategy, use of a different carbamylase as a fusion partner or directed evolution of the enzyme using DNA shuffling is expected to
bypass this barrier (25). Interestingly, the fusion enzymes (CAB-HYD and CAB-HYD1) also catalyzed the conversion of dihydrouracil to -alanine, due to a broad substrate range of both enzymes
(19, 23, 31, 32). It is expected that the fusion enzyme can
be applicable for monitoring the blood level of dihydropyrimidines, an
indicative metabolite accumulated in the dihydropyriminuria that is
caused by a deficiency of dihydropyrimidinase (5, 10).
We have demonstrated the novel ability of microbial
D-hydantoinase to generate a bifunctional fusion enzyme.
Moreover, the functional expression of the bifunctional fusion protein
was readily detected on an activity-staining plate (17). The
results described here raise possibilities that
D-hydantoinase may be effectively used as a novel fusion
partner to construct a fusion protein that is useful for the synthesis
of nonnatural D-amino acids.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Korea Advanced Institute of Science and
Technology, 373-1, Kusung-dong, Yusung-gu, Taejon 305-701, Korea.
Phone: 82-42-869-2616. Fax: 82-42-869-2610. E-mail:
hskim{at}sorak.kaist.ac.kr.
| |
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Abstract
Introduction
