Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas reinhardtii

Article abstract of DOI:10.1016/j.phytochem.2016.12.012

D-Threonine aldolase (DTA) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent interconversion of D-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70?°C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50?°C for 1?h. The enzyme catalyzed the synthesis of D- and D-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of D-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

Full text of DOI:10.1016/j.phytochem.2016.12.012

Phytochemistry xxx (2016) 1e6
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cloning and characterization of -threonine aldolase from the green
D
alga Chlamydomonas reinhardtii
Yuki Hirato a, Mayumi Tokuhisa a, Minoru Tanigawa a, Hiroyuki Ashida b,
, d, *
Hiroyuki Tanaka c, Katsushi Nishimura a
a Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-
8308, Japan
b Department of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Shimane University, Nishikawatsu 1060, Matsue,
Shimane, 690-8504, Japan
c Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Shiga, 520-2192, Japan
d Department of Biotechnology and Materials Chemistry, Junior College, Nihon University, 7-24-1Narashinodai, Funabashi, Chiba, 274-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
D
-Threonine aldolase (DTA) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent interconversion of
threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis
of various -hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga
D-
Received 14 October 2016
Received in revised form
7 December 2016
Accepted 14 December 2016
Available online xxx
b
Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe
the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells;
the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was
purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column
chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and
pH were 70 ꢀC and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 ꢀC for
Keywords:
Chlamydomonas reinhardtii
Chlamydomonadaceae
Green alga
1 h. The enzyme catalyzed the synthesis of
D- and D-allo threonine from a mixture of glycine and
acetaldehyde (the diastereomer excess of -threonine was 18%). DTA was activated by several divalent
D
Enzyme characterization
D
-Amino acid
-Threonine aldolase
metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme
inhibitors.
D
Pyridoxal 5’-phosphate
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
hydroxy-amino acids such as intermediates in the production of
antibiotics and therapeutic drugs for Parkinson's disease (Liu et al.,
D
-Threonine aldolase (DTA, EC 4.1.2.42) catalyzes the intercon-
1999, 2000b). The synthesis reaction leads to the production of
various useful -hydroxy- -amino acids such as active pharma-
ceutical ingredients of drug development candidates (Goldberg
et al., 2015). While glycine is frequently used as the donor mole-
cule, DTA presented in several bacteria, such as Pseudomonas sp.,
version of
b
-hydroxy-
D
-amino acids (e.g.,
D-threonine) and glycine
b
a
plus the corresponding aldehyde. It is pyridoxal 50-phosphate
(PLP)-dependent and classified as a fold-type III enzyme, which
depends on several divalent metal ions (Liu et al., 2000a). DTA
demonstrates high selectivity for
a
-carbon configurations but low
also accepts
D
-alanine and
D-serine (Fesko et al., 2010, 2015).
selectivity for -carbon configurations (Franz and Stewart, 2014).
b
The physiological role of the corresponding
L-threonine aldolase
DTA is a powerful tool for catalyzing CeC bond cleavage and
formation in synthetic organic chemistry (Dückers et al., 2010). The
(LTA, EC 4.1.2.5) is believed to be related to glycine biosynthesis (Liu
et al., 2000a; Franz and Stewart, 2014; Dückers et al., 2010) and LTA
has been found to be necessary in glycine auxotrophic yeast
(McNeil et al., 1994). However, it has been reported that DTA and
LTA are two phylogenetically unique families from different origins,
and the physiological role of DTA is not well understood (Liu et al.,
2015).
cleavage reaction is important for the chiral resolution of
b-
* Corresponding author. Department of Materials and Applied Chemistry, College
of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku,
Tokyo, 101-8308, Japan.
The enzymatic properties of DTA in several bacteria, such as
Arthrobacter sp. DK-38, have been studied in detail (Liu et al.,
E-mail address: nishimura.katsushi@nihon-u.ac.jp (K. Nishimura).
http://dx.doi.org/10.1016/j.phytochem.2016.12.012
0031-9422/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hirato, Y., et al., Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas
reinhardtii, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.012

2
Y. Hirato et al. / Phytochemistry xxx (2016) 1e6
1998; Kataoka et al., 1997). In addition, a mutant alanine race-
mase from Geobacillus stearothermophilus, which shows DTA ac-
tivity, has been developed (Fesko et al., 2008; Seebeck and
Hilvert, 2003). Furthermore, the crystal structure of bacterial
DTA was recently reported, and the enantio-complementarity of
DTA and LTA was explained by the approximate mirror symmetry
of crucial active site residues (Uhl et al., 2015). However, to date,
the existence of eukaryotic DTA has not been reported. In this
study, we report the discovery and characterization of eukaryotic
DTA from the green alga Chlamydomonas reinhardtii. To our
knowledge, this is the first study to report the existence of
eukaryotic DTA.
growth of Chlamydomonas reinhardtii and on the activity of CrDTA
in the green algal cells.
2.2. Purification of recombinant CrDTA
CrDTA was purified to homogeneity from E. coli BL21 (DE3) cells
carrying pCrDTA, with a yield of 3.5% by ammonium sulfate frac-
tionation, and DEAE-Sepharose and Mono Q column chromatog-
raphies (Table 1). The purified enzyme gave a single band with a
molecular mass of 45 kDa on SDS-PAGE (Fig. 2). The N-terminal
amino acid sequence of the protein was determined to be
MRALVSKARLAH. The analyzed N-terminal amino acid sequence
was consistent with that of the deduced sequence from dta. The
molecular mass of the purified enzyme was determined by gel
filtration to be 60 kDa.
2. Results and discussion
2.1. Cloning and sequencing of the gene encoding
aldolase
D-threonine
2.3. Effect of pH and temperature on CrDTA
The sequence of pasa_Sanger_mRNA23075 of Chlamydomonas
reinhardtii, defined as the alanine racemase N-terminal domain,
was obtained from the database at the Chlamydomonas resource
center. The gene encoding DTA from Chlamydomonas reinhardtii
was isolated and sequenced. The open reading frame of 1287 bp
(accession number LC185459) encoded a protein of 428 amino
acids with a calculated molecular mass of 44,999 Da (Fig. 1).
Sequence comparison of the open reading frame with pasa_-
Sanger_mRNA23075 revealed that there was a deletion of 913e948
in the database.
The expression plasmid was constructed from the gene coding
DTA (dta) and pET41b(þ) and the plasmid was transferred into
E. coli BL21 (DE3) cells. However, protein expression was not
detected. As dta contains many rare codons of E. coli, pCrDTA
(dta' þ pET41b(þ)) was used to eliminate the rare codons and was
transferred into E. coli BL21 (DE3) cells. When the recombinant
E. coli was cultured under conditions of addition of 0.2 mM IPTG at
37 ꢀC, the protein was expressed as insoluble inclusion bodies. We
investigated culture conditions (temperature, IPTG concentration,
culture time) that the enzyme was overexpressed as soluble pro-
tein. The cells were grown at 25 ꢀC for 16 h without IPTG, resulting
in the overexpression of a protein possessing DTA activity. The
specific activity of cell-free extract was 0.8 U/mg.
The optimum pH of CrDTA was determined in 0.1 M MES-NaOH
buffer (pH 4.7e7.0), 0.1 M HEPES-NaOH buffer (pH 6.8e8.1), 0.1 M
HEPES-NaOHeNaCl (pH 8.4e8.6), and 0.1 M Bis-Tris-propane-HCl
buffer (pH 8.3e9.0) at 50 ꢀC. The enzyme showed a maximum ac-
tivity at pH 8.4 in 0.1 M HEPES-NaOH buffer (Fig. 3).
The optimum temperature of the enzyme was determined by
measuring its activity in the standard reaction mixture and varying
the temperature from 20 ꢀC to 70 ꢀC. The enzyme showed its
maximum activity at 70 ꢀC (Fig. 4a); however, high temperatures
lager than 70 ꢀC could not be assayed due to the thermal dena-
turation of alcohol dehydrogenase, which was used in the assay.
Seventy percent of its maximum activity was retained between
50 ꢀC and 70 ꢀC.
After incubation of the enzyme at various temperatures for 0, 5,
10, 30, 60, 120, and 180 min, enzyme activity was measured in the
standard reaction mixture at 50 ꢀC. The enzyme showed 90% of
residual activity after 180 min at 50 ꢀC (Fig. 4b). The enzyme began
to be inactivated within 5 min at temperatures greater than 70 ꢀC,
and lost its activity at both 70 ꢀC for 60 min and 80 ꢀC for 5 min.
2.4. Substrate specificity and kinetic parameters of CrDTA
The substrate specificity and kinetic parameters of CrDTA were
Alanine racemase activity has been detected in Chlamydomonas
reinhardtii, but the gene encoding alanine racemase has not been
determined (Nishimura et al., 2007). We predicted that pasa_-
Sanger_mRNA23075 was the gene of Chlamydomonas reinhardtii
alanine racemase because it was annotated as alanine racemase N-
terminal domain. However, the product did not show alanine
racemase activity, but, rather, DTA activity. That was because CrDTA
was similar to alanine racemase as shown below.
We performed amino acid homology search with the primary
sequence of eukaryotic DTA from Chlamydomonas reinhardtii
(CrDTA). CrDTA belongs to the alanine racemase family of PLP en-
zymes (fold-type III). CrDTA was found to be more similar to bac-
terial DTA than to other PLP enzymes (e.g., bacterial alanine
examined with various amino acids (Table 2). The enzyme acted on
b
-hydroxy-
D
-amino acids, such as
D-threonine, D-allo-threonine,
and -threo-phenylserine.
D
D-threo-Phenylserine was the best sub-
strate among the amino acids tested and CrDTA exhibited
35 ꢁ 104
M
ꢂ1sꢂ1 with it. However, inhibition was observed when
-threo-phenylserine exceeded 1 mM. In all
-threo-phenylserine, absorbance at
340 nm decreased linearly. These results, strongly suggested that
inhibition was caused by not product benzaldehyde but substrate
threo-phenylserine. While the enzyme acted on -forms of -hy-
droxy amino acids, the -forms of serine and threonine were inert,
suggesting the enzyme could stereochemically distinguish the
carbon of a substrate but could not distinguish the -carbon.
the concentration of
D
cases of using 1, 3, and 5 mM
D
D-
D
b
L
a
-
b
racemase and eukaryotic D-serine dehydratase). The amino acid
sequence of CrDTA had low sequence identity with bacterial DTAs
(39% and 40% sequence identity with Arthrobacter sp. and Alcali-
genes xylosoxidans, respectively), although bacterial DTA from
Arthrobacter sp. showed high sequence identity (91%) to another
bacterial DTA from A. xylosoxidans. However, the active site residues
were conserved between CrDTA and bacterial DTA from
A. xylosoxidans. Therefore, the physiological role of CrDTA should be
characterized. Our preliminary data indicated that Chlamydomonas
2.5. Synthesis of
D
-threonine and
D
-allo-threonine by CrDTA
CrDTA catalyzed the synthesis of
nine from glycine and acetaldehyde. The enzymatic product
comprised a mixture of -threonine and -allo-threonine. After 1 h
incubation, conversion rates of -threonine and -allo-threonine
were 6.7 and 4.6%, respectively, and the diastereomer excess of
threonine was determined to be 18%. Specific activities of synthesis
for -threonine and -allo-threonine were 50.9 and 36.8 mol/min/
mg, respectively. The ratio of conversion rate of -threonine to that
D-threonine and D-allo-threo-
D
D
D
D
D
-
reinhardtii exhibited
D
-threonine aldolase activity, and we are
-threonine on
D
D
m
presently attempting to characterize effects of
D
D
Please cite this article in press as: Hirato, Y., et al., Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas
reinhardtii, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.012

Fig. 1. Nucleotide and deduced amino acid sequences of dta and dta'. The asterisk denotes a translational stop codon. The underlined amino acid sequence is identical to that was
determined by Edman degradation for the purified enzyme.
Please cite this article in press as: Hirato, Y., et al., Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas
reinhardtii, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.012

4
Y. Hirato et al. / Phytochemistry xxx (2016) 1e6
Table 1
Purification of CrDTA from E. coli BL21 (DE3) harboring pCrDTA.
Step
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Purification (fold)
Yield (%)
Cell-free extract
Ammonium sulfate
DEAE-Sepharose
Mono Q
4520
2750
482
5490
3280
115
0.823
0.840
4.20
1.00
1.02
5.11
8.56
100
60.9
10.7
3.51
159
22.5
7.05
Threonine aldolase activity was determined using D-threonine as substrate.
threonine was a cause of diastereomer excess in the enzymatic
synthesis.
2.6. Effect of various compounds on CrDTA
CrDTA was activated by MnCl2, CoCl2, NiCl2, and MgCl2, but was
strongly inhibited by CaCl2, CuCl2, and ZnCl2 (all 100 mM) (Table 3).
In addition, the metalloenzyme inhibitors (EDTA and sodium cya-
nide) strongly inhibited the enzyme activity. These results
demonstrate that CrDTA requires particular divalent metals as co-
factors. The enzyme reaction was also strongly inhibited by hy-
droxylamine (Table 3). Moreover, at pH 8.0, the enzyme showed
maximum absorptions at 280 and 416 nm (data not shown). This
inhibition and the absorption spectrum indicate that the enzyme
requires PLP as a coenzyme. Thiol reagents, iodoacetate and p-
chloromercuribenzoic acid, and the chelating agent 8-
hydroxyquinoline had no effect on the enzyme reaction.
These results indicating the metal dependency of CrDTA agreed
well with bacterial DTA from Arthrobacter sp. DK-38 (Kataoka
et al., 1997). The zinc ion in eukaryotic D-serine dehydratase is a
five coordination (Ito et al., 2008), and the copper ion is a skewed
octahedron. In this study, the enzyme was activated in the pres-
ence of divalent metal ions which are regular octahedrons. We
considered that the divalent metal ion activating CrDTA was
stringently controlled by the coordinate bond of CrDTA active site
residues. Then, invasion of 8-hydroxyquinoline might be pre-
vented by the stringently controlled metal binding site, resulting
in no effect as mentioned above. The crystal structure of bacterial
DTA from A. xylosoxidans identified metal-binding residues in the
active site (Uhl et al., 2015). The observation was almost consistent
with our thought about metal coordination for CrDTA. X-ray
crystallography of CrDTA-substrate complex is currently being
examined to clarify the residues that interact with substrate, co-
enzyme, and metal ion.
Fig. 2. Analysis of the expression and purification of CrDTA by SDS-PAGE. Cell-free
extract (lane 1), active fractions from ammonium sulfate fractionation (lane 2), DEAE-
Sepharose (lane 3), and Mono Q (lane 4), and protein marker (lane M) for calibration.
Protein bands were detected by Coomassie Brilliant Blue staining.
Bacterial DTA has been shown to be strongly inhibited by thiol
reagents (Kataoka et al., 1997); however, these agents had no effect
on CrDTA. Amino acid sequence comparison between CrDTA and
bacterial DTA indicated that the cause of inhibition was likely the
cysteine 303 of bacterial DTA in the vicinity of the active site. In the
case of CrDTA, this position is occupied by a serine residue at 329.
3. Materials and methods
3.1. Materials
Microalgal strain Chlamydomonas reinhardtii NIES-2237 was
obtained from the National Institute for Environmental Studies
(Ibaraki, Japan).
D-threo-Phenylserine was kindly provided by
Fig. 3. Effect of pH on CrDTA activity using 0.1 M MES-NaOH buffer (pH 4.7e7.0, open
circle), 0.1 M HEPES-NaOH buffer (pH 6.8e8.1, open square), 0.1 M Bis-Tris-propane-
HCl buffer (pH 8.3e9.0, open triangle), 0.1 M HEPES-NaOHeNaCl buffer (pH 8.4e8.6,
closed circle). Activity was expressed relative to that determined at pH 8.4 (100%).
Associate Professor Hisashi Muramatsu (Kochi University). NADH,
yeast alcohol dehydrogenase, and molecular marker proteins for
gel filtration were obtained from Oriental Yeast (Tokyo, Japan).
Restriction enzymes for genetic manipulation were obtained from
Takara Bio (Siga, Japan). Plasmids were obtained from Merck Mil-
lipore (Massachusetts, USA) and GenScript (New Jersey, USA). Ge-
netic manipulation kits were obtained from Qiagen (Hilden,
of
threonine to that with
difference between specific activities with
D
-allo-threonine was 1.46, and the ratio of specific activity with
-allo-threonine was 1.38. Therefore, the
-threonine and -allo-
D-
D
D
D
Please cite this article in press as: Hirato, Y., et al., Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas
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Y. Hirato et al. / Phytochemistry xxx (2016) 1e6
5
Fig. 4. (a) Effect of temperature on CrDTA activity. Activity was expressed relative to that determined at 70 ꢀC (100%). (b) Thermostability of DTA at 50 ꢀC (open circle), 70 ꢀC (open
square), and 80 ꢀC (open triangle). Activity was expressed as residual activity remaining when compared to using enzyme before incubation (100%).
Table 2
Briefly, the assay mixture comprised 100 mM HEPES-NaOH (pH
8.1), 5 mM D-threonine, 50 mM PLP, 100 mM MnCl2, 200 mM NADH,
Substrate specificity and kinetic parameters of CrDTA.
26 U/mL yeast alcohol dehydrogenase, and appropriate amounts of
CrDTA solution in a final volume of 0.5 mL. One unit of the enzyme
was considered the amount of enzyme thatþcatalyzed the formation
of 1 mmol of acetaldehyde (1 m
mol of NAD ) per min at 50 ꢀC; the
Substrate
Relative
activity (%)
Km
(mM)
kcat
kcat/Km
(sꢂ1
)
(Mꢂ1sꢂ1
)
D
D
-Threonine
-allo-Threonine
100
406
ND
ND
1230
ND
0.31
2.4
e
4.2
25
e
1.3 ꢁ 104
1.1 ꢁ 104
molar extinction coefficient of NADH is 6.2 ꢁ 103 Mꢂ1 cmꢂ1
.
L
L
-Threonine
-allo-Threonine
e
e
e
e
Aldolase activity toward phenylserine was measured spectropho-
tometrically at 280 nm; the molar extinction coefficient of pro-
duced benzaldehyde is 1.4 ꢁ 103 Mꢂ1 cmꢂ1. Control experiments
were carried out under the same conditions without the addition of
enzyme solution.
D
-threo-Phenylserine
0.81
e
280
e
35 ꢁ 104
D
-Serine
e
e
L
-Serine
ND
e
e
ND, not detectable.
The ability of CrDTA to synthesize D-threonine and D-allo-thre-
Germany), Takara Bio, and Toyobo (Osaka, Japan). Oligonucleotide
primers were synthesized by Fasmac (Kanagawa, Japan). All other
reagents of analytical grade were obtained from Sigma (St. Louis,
USA), Nacalai Tesque (Kyoto, Japan), and Wako Pure Chemical In-
dustries (Osaka, Japan). HiTrap DEAE FF, Mono Q 5/50 GL, and
Superose 12 10/300 GL were purchased from GE Healthcare UK Ltd.
(Buckinghamshire, England). The Cosmosil 5C18-AR-300 packed
column was purchased from Nacalai Tesque.
onine was assayed by high performance liquid chromatography
(HPLC). We modified the synthetic activity analysis method as
described previously (Kataoka et al., 1997). Briefly, the reaction
mixture, composed of 100 mM HEPES-NaOH (pH 8.1), 100 mM
glycine, 350 mM acetaldehyde, 10
mM PLP, 1 mM MnCl2, and
appropriate amounts of the enzyme solution in a final volume of
0.5 mL, was incubated at 50 ꢀC for 0, 15, 30, or 60 min, and termi-
nated with 0.5 mL of 1 M HCl. After appropriate dilution and
filtration, the synthesized amino acids were derivatized with
derivatization reagent. To prepare the derivatization reagent, 15 mg
of o-phthaldialdehyde was dissolved in 0.5 mL ethanol and mixed
with 11 mL of 0.1 M sodium borate buffer (pH 10) and 30 mg N-
3.2. Enzyme assays
Threonine aldolase activity was spectrophotometrically
measured at 340 nm by coupling the reduction of acetaldehyde
(oxidation of NADH) with yeast alcohol dehydrogenase. We modi-
fied the coupling method as described previously (Liu et al., 1998).
acetyl-
reagent was added to 100
derivatized sample (20
produced -threonine and
and optical purities of -threonine and
L
-cysteine. Three-hundred-microliters of this derivatization
L of the sample solution. A portion of the
L) was used to determine the amount of
-allo-threonine. The concentrations
-allo-threonine were
m
m
D
D
D
D
Table 3
analyzed using an HPLC system equipped with a Cosmosil 5C18-AR-
300 packed column (4.6 ꢁ 250 mm) maintained at 30 ꢀC. Deriv-
atized samples were applied and eluted with a linear gradient of
22e25% methanol in 50 mM sodium acetate buffer (pH 6.5). The
Effect of various compounds on the activity of CrDTA.
Compounds
Nonea
Concentration (mM)
Relative activity (%)
40.8
100
87.5
83.6
75.7
13.3
0
0
5.2
89.2
93.2
105
0
a
MnCl2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
1
1
1
1
1
flow rate was 0.7 mL/min, and elution times for
D-threonine and D-
a
CoCl2
NiCl2
a
allo-threonine were 8.5 and 18.5 min, respectively.
a
MgCl2
a
CaCl2
3.3. Isolation and sequencing of the gene encoding
aldolase from Chlamydomonas reinhardtii
D-threonine
a
CuCl2
ZnCl2
a
Hydroxylamineb
Iodoacetateb
Total RNA was isolated using the RNeasy Plant Mini Kit (QIA-
GEN) with Chlamydomonas reinhardtii NIES-2237. Based on the
cording sequence in the database of the Chlamydomonas resource
center (Chlre4, http://www.chlamycollection.org/), the oligonu-
cleotide primers, 50-ATGCGGGCGCTGGTTTCC-30 as the sense
primer and 50-TCATTGCCCTGGCCCCCGCCC-30 as the antisense
primer, were designed. Reverse transcription was performed with
p-Chloromercuribenzoic acidb
8-Hydroxyquinolineb
EDTAb
Sodium cyanideb
1
12.7
a
Effect of metal salts on the activity was assayed using standard reaction mixture
except for MnCl2.
b
Effect of inhibitors was tested with reaction mixture containing MnCl2.
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6
Y. Hirato et al. / Phytochemistry xxx (2016) 1e6
ReverTra Ace (Toyobo), the antisense primer, and total RNA of
Chlamydomonas reinhardtii NIES-2237. Amplification of nucleotides
between the two primers was performed using PCR with KOD-
plus- Ver. 2 (Toyobo) and the reverse transcription product as the
template. The amplified product was purified using SUPREC-PCR
(Takara Bio). Nucleotide sequencing was analyzed using a genetic
analyzer 3100xl equipped with a Big Dye Terminator ver. 3.1
(Applied Biosystems). We named the gene encoding DTA dta.
Nucleotide and amino acid sequences were obtained from the
database at the Chlamydomonas resource center and NCBI (www.
ncbi.nlm.nih.gov/). A homology search was performed with the
BLAST program (Altschul et al., 1990) at NCBI. Multiple alignments
were performed with DNAMAN 6.0 (Lynnon LLC; CA, USA). Kinetic
parameters (kcat and Km) were calculated according to Lineweaver-
Burk plots.
References
3.4. Expression and purification of D-threonine aldolase in E. coli
BL21 (DE3)
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local
alignment search tool. J. Mol. Biol. 215, 403e410.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 72, 248e254.
Based on genetic information revealed from mRNA, the
expression plasmid pCrDTA was constructed from the gene
encoding DTA with codons optimized for expression in E. coli (dta',
Fig. 1) and pET-41b(þ). The pCrDTA was transferred into chemically
competent E. coli BL21 (DE3) cells using the heat shock method to
produce recombinant DTA. Twenty-four-milliliters of the trans-
formant, which was precultured at 37 ꢀC overnight, was inoculated
into 1.2 L of LB medium (in a 2 L Marineflask, Furukawa riko, Tokyo,
€
Dückers, N., Baer, K., Simon, S., Groger, H., Hummel, W., 2010. Threonine aldola-
sesdscreening, properties and applications in the synthesis of non-
proteinogenic
b
-hydroxy-
a
-amino acids. Appl. Microbiol. Biotechnol. 88,
409e424.
Fesko, K., Giger, L., Hilvert, D., 2008. Synthesis of
b
-hydroxy- -amino acids with a
a
reengineered alanine racemase. Bioorg. Med. Chem. Lett. 18, 5987e5990.
Fesko, K., Uhl, M., Steinreiber, J., Gruber, K., Griengl, H., 2010. Biocatalytic Access to
a, a-dialkyl-a-amino acids by a mechanism-based approach. Angew. Chem. Int.
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Japan) containing 25 m
g/mL kanamycin. Cells were grown at 25 ꢀC
Fesko, K., Strohmeier, G.A., Breinbauer, R., 2015. Expanding the threonine aldolase
for 16 h and harvested by centrifugation. E. coli cells harboring
pCrDTA were suspended in 100 mM Tris-HCl buffer (pH 8.0) con-
taining 50 mM PLP and 1 mM phenylmethylsulfonyl fluoride, and
disrupted by ultrasonication on ice. After centrifugation, the cell-
free extract was fractionated by ammonium sulfate precipitation.
The active fraction (25e60% saturation) was resuspended in 50 mM
toolbox for the asymmetric synthesis of tertiary a-amino acids. Appl. Microbiol.
Biotechnol. 99, 9651e9661.
Franz, A.E., Stewart, J.D., 2014. Threonine aldolases. Adv. Appl. Microbiol. 88,
58e101.
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Hang, C., Rossano, L.T., Schmidt, M.A., 2015. Preparation of b-hydroxy-a-amino
acid using recombinant d-threonine aldolase. Org. Process Res. Dev. 19,
1308e1316.
Tris-HCl buffer (pH 8.0) containing 20 mM PLP and dialyzed against
Ito, T., Hemmi, H., Kataoka, K., Mukai, Y., Yoshimura, T., 2008. A novel zinc-
the same buffer. The enzyme solution was centrifuged and filtered
to remove particulates, applied to ten tandemly connected Hitrap
DEAE FF columns equilibrated with 50 mM Tris-HCl buffer (pH 8.0)
dependent
D-serine dehydratase from Saccharomyces cerevisiae. Biochem. J.
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D-threonine aldolase, a
containing 20
mM PLP, and eluted with a linear gradient of
pyridoxal-5’-phosphate-dependent enzyme from Arthrobacter sp. DK-38. Eur. J.
Biochem. 248, 385e393.
0e500 mM NaCl in the same buffer at a flow rate of 1 mL/min.
Active fractions were pooled and dialyzed against 50 mM Tris-HCl
Liu, G., Zhang, M., Chen, X., Zhang, W., Ding, W., Zhang, Q., 2015. Evolution of
threonine aldolases, a diverse family involved in the second pathway of glycine
biosynthesis. J. Mol. Evol. 80, 102e107.
buffer (pH 8.0) containing 20
solution was then applied to a Mono Q 5/50 GL column equilibrated
with 50 mM Tris-HCl buffer (pH 8.0) containing 20 M PLP and
mM PLP. After filtration, the enzyme
Liu, J.Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S., Yamada, H., 1998. A novel metal-
m
activated pyridoxal enzyme with a unique primary structure, low specificity D-
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16678e16685.
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threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of
antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed ste-
reospecific resolution. Appl. Microbiol. Biotechnol. 51, 586e591.
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microbial threonine aldolases and their application. J. Mol. Catal. B Enzym. 10,
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eluted with a linear gradient of 0e500 mM NaCl in the same buffer
at a flow rate of 0.5 mL/min. The active fractions were pooled and
stored at ꢂ80 ꢀC until use.
3.5. Other analytical methods
Estimation of the molecular mass was performed using Super-
ose 12 column chromatography with the following protein
markers: glutamate dehydrogenase (290 kDa), enolase (67 kDa),
myokinase (32 kDa), and cytochrome c (12.4 kDa). The protein
concentration was determined by the Bradford method (Bradford,
1976) with bovine serum albumin as the standard. The purity and
molecular mass of each subunit of the enzyme were examined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) with the CLEARLY Protein Ladder (Takara Bio). Gels were
stained with Quick Blue Staining Solution (BioDynamics Laboratory
Inc., Tokyo, Japan). The N-terminal amino acid sequence of the
enzyme was determined by the Edman degradation procedure with
a Procise 492 cLC protein sequencer (Applied Biosystems, CA, USA)
Liu, J.Q., Odani, M., Yasuoka, T., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S., Yamada, H.,
2000b. Gene cloning and overproduction of low-specificity
D-threonine
aldolase from Alcaligenes xylosoxidans and its application for production of a
key intermediate for parkinsonism drug. Appl. Microbiol. Biotechnol. 54, 44e51.
McNeil, J.B., McIntosh, E.M., Taylor, B.V., Zhang, F., Tangs, S., Bognar, A.L., 1994.
Cloning and molecular characterization of three genes, including two genes
encoding serine hydroxymethyltransferases, whose inactivation is required to
render yeast auxotrophic for Glycine. J. Biol. Chem. 269, 9155e9165.
Nishimura, K., Tomoda, Y., Nakamoto, Y., Kawada, T., Ishii, Y., Nagata, Y., 2007.
Alanine racemase from the green alga Chlamydomonas reinhardtii. Amino acids
32, 59e62.
Seebeck, F.P., Hilvert, D., 2003. Conversion of a PLP-dependent racemase into an
aldolase by a single active site mutation. J. Am. Chem. Soc. 125, 10158e10159.
Uhl, K.K., Oberdorfer, G., Steinkellner, G., Berket, L.R., Mink, D., Assema, F.,
Schürmann, M., Gruber, K., 2015. The crystal structure of D-threonine aldolase
from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP
dependent mechanism. PLoS One 10, 1e15.
and
a PPSQ-10 protein sequencer (Shimadzu, Kyoto, Japan).
Please cite this article in press as: Hirato, Y., et al., Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas
reinhardtii, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.012