A Novel Metal-activated Pyridoxal Enzyme with a Unique Primary
Structure, Low Specificity D-Threonine Aldolase from
Arthrobacter sp. Strain DK-38
MOLECULAR CLONING AND COFACTOR CHARACTERIZATION*
Ji-Quan
Liu
,
Tohru
Dairi
,
Nobuya
Itoh
,
Michihiko
Kataoka§,
Sakayu
Shimizu§, and
Hideaki
Yamada
¶
From the
Laboratory of Biocatalytic Chemistry,
Biotechnology Research Center, Toyama Prefectural University,
Kosugi Machi, Toyama 939-0398 and § Division of Applied
Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto
606-8502, Japan
 |
ABSTRACT |
The gene encoding low specificity
D-threonine aldolase, catalyzing the interconversion
of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of
Arthrobacter sp. strain DK-38. The gene contains an open
reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant
Escherichia coli cells and purified to homogeneity by
ammonium sulfate fractionation and three-column chromatography steps.
The recombinant aldolase was identified as a pyridoxal enzyme with the
capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit,
and Lys59 of the enzyme was determined to be the cofactor
binding site by chemical modification with NaBH4. In
addition, Mn2+ ion was demonstrated to be an activator of
the enzyme, although the purified enzyme contained no detectable metal
ions. Equilibrium dialysis and atomic absorption studies revealed that
the recombinant enzyme could bind 1 mol of Mn2+ ion per mol
of subunit. Remarkably, the predicted amino acid sequence of the enzyme
showed no significant similarity to those of the currently known
pyridoxal 5'-phosphate-dependent enzymes, indicating that
low specificity D-threonine aldolase is a new pyridoxal
enzyme with a unique primary structure. Taken together, low specificity
D-threonine aldolase from Arthrobacter sp.
strain DK-38, with a unique primary structure, is a novel
metal-activated pyridoxal enzyme.
 |
INTRODUCTION |
The bioorganic synthesis of
-hydroxy-
-amino acids attracts a
great deal of attention because of their potential application as
chiral building blocks for the synthesis of biologically active molecules (1-5). A variety of
-hydroxy-
-amino acids is present in complex natural compounds with interesting biological properties. 3,4,5-Trihydroxy-L-aminopentanoic acid is a key component
of polyoxins (1). 4-Hydroxy-L-threonine, for example, is a
precursor of rizobitoxine, a potent inhibitor of pyridoxal 5'-phosphate
(PLP)1-dependent
enzymes (1). The D-isomers are also biologically significant, because they not only exist in mature mammals (6) but are
also constituents of a range of antibiotics, for example, Fusaricidin (7) and Viscosin (8).
Threonine aldolase (TA) (EC 4.1.2.5), which catalyzes the reversible
interconversion of certain
-hydroxy-
-amino acids and glycine plus
aldehydes, has been shown to be useful for the biosynthesis of
-hydroxy-
-amino acids (1-5). The enzyme appears to fall into two
types, L-type and D-type, on the basis of its stereospecificity of the cleavage reaction toward the
-carbon of
threonine. L-Type TA, acting on L- and/or
L-allo-threonine, is further divided into three
groups based on its stereospecificity toward the
-carbon of
threonine as follows: (i) L-allo-TA is specific
to L-allo-threonine; (ii) L-TA acts
only on L-threonine; and (iii) low specificity
L-TA can use both L-threonine and
L-allo-threonine as substrates. All of the three
L-type enzymes have been found to exist in nature.
L-TA was partially purified from Clostridium pasteurianum (9); L-allo-TA was from
Aeromonas jandaei (10), and three low specificity
L-TAs from Candida humicola (11, 12), Saccharomyces cerevisiae (13), and Pseudomonas
sp. strain (14) were purified and extensively characterized. The genes
encoding for L-allo-TA of A. jandaei
and low specificity L-TAs of S. cerevisiae and
Pseudomonas sp. strain have been cloned and sequenced
(13-15). Likewise, D-type TA, acting on
D-threonine and/or D-allo-threonine, might include D-allo-TA, D-TA, and
low specificity D-TA. However, only low specificity
D-TA activity was found from 3 out of 2,000 strains
examined (16). Low specificity D-TA was previously purified from Arthrobacter sp. strain DK-38, and the enzyme was shown
to have a monomeric structure and to require unusually both PLP and divalent cations for its catalytic activity (16). Due to little available purified enzyme, the identification of the cofactors was not
performed.
The present paper describes cloning and sequencing of the
dtaAS gene encoding the low specificity D-TA
from Arthrobacter sp. strain DK-38, the expression of the
gene in Escherichia coli cells, and further characterization
of the recombinant enzyme. Our data showed that the low specificity
D-TA is a novel metal-activated pyridoxal enzyme. Evidence
is also presented that low specificity D-TA has a unique
primary structure, probably representing a new family of pyridoxal
enzyme.
 |
EXPERIMENTAL PROCEDURES |
Materials
Butyl-Toyopearl 650M and DEAE-Toyopearl 650M were purchased from
Tosoh (Tokyo, Japan); Gigapite was obtained from Toagosei (Tokyo,
Japan). Pyridoxal-5'-phosphate (PLP) and pyridoxime-5'-phosphate monohydrochloride were bought from Nacalai (Kyoto, Japan).
DL-threo-
-Phenylserine and
DL-threo-
-(3,4-dihydroxyphenyl)serine were
purchased from Sigma.
DL-erythro-
-Phenylserine was a generous gift
from Hideyuki Hayashi and Hiroyuki Kagamiyama, Department of
Biochemistry, Osaka Medical College, Osaka, Japan.
DL-threo- and
DL-erythro-
-(3,4-methylenedioxyphenyl)serine were prepared according to the method of Ohashi et al. (17). The other chemicals were all analytical grade reagents.
Bacterial Strains, Plasmids, and Culture Conditions
Arthrobacter sp. strain DK-38 was used as the source
of chromosomal DNA (16). E. coli XL1-Blue MRF' (recA1
thi endA1 supE44 gyrA46 relA1 hsdR17 lac/F'
[proAB+ lacIq
lacZ_M15::Tn10 Tetr]) (Toyobo,
Osaka, Japan) was used as a host for the gene cloning. Plasmid pUC118
(Takara Shuzo, Kyoto, Japan) was used as a cloning vector. pKK 223-3 (Amersham Pharmacia Biotech, Uppsala, Sweden) was used as a vector for
overexpression of the dtaAS gene. Arthrobacter sp. strain DK-38 was grown in a medium comprising 1% peptone, 1%
yeast extract, and 0.5% NaCl (pH 7.2). Recombinant E. coli cells were cultivated at 37 °C in Luria-Bertani (LB) medium (1% peptone, 0.5% yeast extract, and 1% NaCl (pH 7.2)) containing 0.1 mg/ml ampicillin. For induction of the gene under the control of the
lac or tac promoter, 0.2 mM
isopropyl-
-D-thiogalactoside (IPTG) was added to the LB
medium.
General Recombinant DNA Technique
Plasmid DNA was purified with a plasmid purification kit from
Qiagen, Inc. (Chatsworth, CA). Restriction enzymes and DNA-modifying enzymes were purchased from Takara Shuzo and Toyobo and used according to the manufacturers' protocols. Transformation of E. coli
with plasmid DNA by electroporation was performed under standard
conditions with a BTX ECM 600 electroporation system (Biotechnologies
and Experimental Research, Inc., San Diego, CA). Other general
procedures were performed as described by Sambrook et al.
(18).
Cloning of the dtaAS Gene
Two oligonucleotide primers were purchased from Amersham
Pharmacia Biotech (Tokyo, Japan), each with additional restriction sites (underlined in the following sequences) added to the 5' end to
facilitate cloning of the amplified product: primer I, 5'-CCGAAGCTTATGTCNCARGARGTNAT-3'; and primer II,
5'-GCCGAATTCGGGSGTGTCNACNCGSGC-3'. Degenerate positions are
indicated by "S" for C or G, "R" for A or G, and "N" for
all bases. Primers I and II were based upon the
NH2-terminal amino acid sequence of the wild-type low
specificity D-TA from Arthrobacter sp. strain
DK-38. PCR amplification was performed in a 50-µl reaction mixture
containing 5 µl of Me2SO, 10 mM Tris-HCl (pH
8.85), 25 mM KCl, 5 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1 mM each deoxynucleotide
triphosphate, 20 pmol of each primer, 1 µg of the genomic DNA, and
0.5 units of PWO DNA polymerase (Boehringer Mannheim,
Germany) at 94 °C for 1 min, 43 °C for 0.5 min, and 72 °C for
1 min for a total of 35 cycles. The amplified product was digested with
EcoRI and HindIII, separated by agarose gel electrophoresis, and then purified with a GeneClean kit (Bio 101, Vista, CA). The amplified DNA of 90 bp was then cloned into pUC118.
Chromosomal DNA isolated from Arthrobacter sp. strain DK-38
cells was partially digested with Sau3AI and fractionated on
a sucrose density gradient (10-40%) in a Beckman L-70 ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA) at 100,000 × g for 18 h. Fragments in the molecular size of 1-10 kb
were collected and ligated into BamHI-restricted pUC118, and
the plasmids were introduced into E. coli XL1-Blue MRF'
cells to construct a genomic library of Arthrobacter sp.
strain DK-38. The genomic library was screened by colony hybridization
with the [
-32P] dATP-labeled 90-bp DNA fragment as a
probe. The clone, pUDTA, carrying an approximately 7.2-kb DNA fragment
was selected for further analysis.
Nucleotide Sequence Analysis
pUDTA was used as a sequencing template. The nucleotide sequence
was determined by the dideoxy chain termination method with Cy5
AutoRead and Cy5 AutoCycle sequencing kits and an Amersham Pharmacia Biotech ALFred DNA sequencer. A homology search was performed
by the sequence similarity searching programs Fasta (19) and Blast
(20). The ClustalW method was used to align the sequences (21).
Overexpression of the dtaAS Gene in E. coli Cells
To obtain the entire gene without excessive flanking parts, PCR
amplification was carried out in a 50-µl reaction mixture containing
5 µl of Me2SO, 10 mM Tris-HCl (pH 8.85), 25 mM KCl, 5 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1 mM deoxynucleotide triphosphate, 20 pmol of each primer, 1 µg of the genomic DNA, and 0.5 units of
PWO DNA polymerase (Boehringer Mannheimn) at 94 °C for 1 min, 60 °C for 2 min, and 72 °C for 3 min for a total of 30 cycles. The 5' primer containing a Shine-Dalgarno sequence (lowercase letters) and an ATG initiation codon (bold letters), and the 3' primer
with the complement of the TGA termination codon (bold letters) had the
respective sequences
5'-GCCGAATTCggagCGTCCCGATGTCCCAGG-3' and
5'-CCGGAATTCTGAAGACGTCAGCGCGAG-3', which were
designed on the basis of the nucleotide sequence of the
dtaAS gene; to facilitate the cloning, an additional
restriction site (underlined sequence) was incorporated into both
primers. The amplified PCR product was digested with EcoRI,
separated by agarose gel electrophoresis, and then purified with a
GeneClean kit. The amplified DNA of approximately 1.1 kb, which
contained the complete coding sequences of the dtaAS gene,
was inserted downstream of the tac promoter in pKK223-3 and
then used to transform E. coli XL1-Blue MRF' cells. The
constructed plasmid was designated pKDTA.
Purification of the Recombinant Low Specificity
D-TA
All enzyme purification operations were carried out at
0-4 °C. Unless otherwise noted, 50 mM Tris-HCl (pH 7.4)
containing 10 µM PLP was used as the buffer throughout
the purification procedures.
Step 1, Preparation of Cell Extract--
Cells of the E. coli transformant harboring plasmid pKDTA were grown aerobically
at 37 °C for 16 h in 12 liters of LB medium containing 0.1 mg/ml ampicillin and 0.2 mM IPTG. The cells were harvested
and rinsed with buffer. After being suspended in 200 ml of buffer, the
cells were disrupted by ultrasonic oscillation at 4 °C for 20 min
with a model 201M ultrasonic oscillator (Kubota, Tokyo, Japan). The
cell debris was removed by centrifugation at 25,000 × g for 30 min.
Step 2, Ammonium Sulfate Fractionation--
The supernatant
solution was brought to 20% saturation with ammonium sulfate and
centrifuged at 25,000 × g for 30 min. Ammonium sulfate
was added to the supernatant solution to 50% saturation. The
precipitate collected by centrifugation was dissolved in buffer, and
the enzyme solution was dialyzed against 1,000 volumes of buffer.
Step 3, Butyl-Toyopearl Column Chromatography--
The enzyme
solution, brought to 20% saturation with ammonium sulfate, was applied
to a Butyl-Toyopearl 650M column (5.0 × 40 cm). Elution was
carried out with a 2,400-ml linear gradient of 20 to 0% saturated
ammonium sulfate in buffer at a flow rate of 5 ml/min. The fractions
with threonine aldolase activity were pooled and concentrated by
ultrafiltration with a Centriprep-30 apparatus (Amicon, Inc., Beverly,
MA).
Step 4, DEAE-Toyopearl Column Chromatography--
The enzyme
solution was dialyzed against 1,000 volumes of buffer and applied to a
DEAE-Toyopearl 650 M column (2.5 × 20 cm) equilibrated with buffer. After the column was thoroughly washed with
the buffer containing 50 mM NaCl, linear gradient elution was performed with a buffer supplemented with NaCl by increasing the
concentration from 50 to 200 mM. The flow rate was
maintained at 5 ml/min. The fractions with D-TA activity
were pooled and concentrated by ultrafiltration with a Centriprep-30
apparatus.
Step 5, Gigapite Column Chromatography--
The enzyme solution
was dialyzed against 1,000 volumes of 10 mM potassium
phosphate buffer (pH 7.4) containing 10 µM PLP and applied to a Gigapite column (5.0 by 40 cm) equilibrated with the 10 mM potassium phosphate buffer (pH 7.4). After the column was thoroughly washed with the same buffer, elution was carried out
with a 2,400-ml linear gradient of 10-300 mM potassium
phosphate buffer (pH 7.0) at a flow rate of 5 ml/min. The fractions
with D-TA activity were pooled and concentrated by
ultrafiltration with a Centriprep-30 apparatus. The purified enzyme was
concentrated to a protein concentration of about 25 mg/ml and stored at
0 to 4 °C for at least 1 month without loss of enzyme activity.
Amino Acid Sequencing
Amino acid sequence was determined by the Edman degradation
procedure with a model 476A protein sequencer (Perkin-Elmer) or a model
PPSQ-10 protein sequencer (Shimadzu, Kyoto, Japan).
Enzyme Assay
Threonine aldolase activity was measured spectrophotometrically
at 340 nm by coupling the reduction of acetaldehyde (oxidation of NADH)
with yeast alcohol dehydrogenase (Wako, Osaka, Japan). The assay
mixture comprised 100 µmol of Hepes-NaOH buffer (pH 8.0), 50 µmol
of D-threonine, 0.05 µmol of PLP, 0.1 µmol of
MnCl2, 0.2 µmol of NADH, 30 units of yeast alcohol
dehydrogenase, and appropriate amounts of the enzyme in a final volume
of 1 ml. One unit of the enzyme is the amount of enzyme that catalyzed
the formation of 1 µmol of acetaldehyde (1 µmol of NADH oxidized) per min at 30 °C; the molar extinction coefficient of NADH is 6.2 × 103 M
1
cm
1. For a qualitative analysis, threonine aldolase
activity was also assayed as follows: the reaction mixture comprised 10 µmol of D-threonine, 0.01 µmol of PLP, 0.02 µmol of
MnCl2, 20 µmol of Hepes-NaOH buffer (pH 8.0), and the
enzyme in a total volume of 200 µl. The reaction was carried out at
30 °C for 10 min and was terminated by the addition of 50 µl of
30% trichloroacetic acid. The released acetaldehyde was measured
spectrophotometrically according to the method of Paz et al.
(22). The aldolase activities toward phenylserine,
-3,4-dihydroxyphenylserine, and
-3,4-methylenedioxyphenylserine were measured spectrophotometrically at 279, 350, and 320 nm, respectively, with molar extinction coefficients of 1.4 × 103 M
1 cm
1 for
benzaldehyde, 8.9 × 103 M
1
cm
1 for protocatechualdehyde, and 17.6 × 103 M
1 cm
1 for
piperonal.
Determination of Protein
The concentration of the enzyme was determined
spectrophotometrically by using a molar extinction coefficient
M = 29,927 M
1
cm
1 (A1 cm1 mg/ml = 1.34) at 278 nm and pH 7.4 for the holoenzyme and
M = 27,580 M
1 cm
1
(A1 cm1 mg/ml = 1.45) at 278 nm
for the apoenzyme, where the
M values were determined by
the method of Edelhoch and co-workers (23, 24), and the contributions
of tryptophan, tyrosine, and cystine to the
M values in
6 M guanidine hydrochloride were calculated on the basis of
3 tryptophan, 7 tyrosine, and 8 cystine residues and 5565, 1395, and
140 M
1 cm
1 for the three
residues, respectively.
Chromatographic Optical Resolution of Amino Acid
Stereoisomers
The isomers of
-phenylserine and
-(3,4-methylenedioxyphenyl)serine were analyzed by high
performance liquid chromatography as follows: column, Sumichiral
OA-5000 (4.6 × 150 mm) (Sumitomo, Tokyo, Japan); solvent, 2 mM copper sulfate containing 15% methanol; flow rate, 1.0 ml/min; detection, 254 nm; and temperature, 30 °C. The isomers of
-(3,4-dihydroxyphenyl)serine were also analyzed by high performance
liquid chromatography as follows: column, Crownpak CR (
) (4 × 150 mm) (Daicel, Osaka, Japan); solvent, distilled water adjusted to pH
1.0 with perchloric acid; flow rate, 0.4 ml/min; detection, 220 nm; and
temperature, 4 °C.
Spectrophotometric Measurements
The absorption spectrum of the enzyme was measured at 20 °C
with 20 mM potassium phosphate buffer (pH 7.4) by a Hitachi
model U-3210 spectrophotometer (Hitachi, Tokyo, Japan).
PLP Content
The PLP content of the enzyme was determined according to the
method of Wada and Snell (25).
Isolation of Pyridoxyl Peptide
The holoenzyme (10 mg in 200 µl of 20 mM potassium
phosphate buffer (pH 7.4)) was reduced at 0 °C by the addition of
about 0.2 mg of solid NaBH4. To protect the label against
photo-destruction, all the tubes used in the following experiment were
covered with aluminum foil. After standing at 0 °C for about 30 min,
the enzyme protein was precipitated with 2% trichloroacetic acid. The
precipitates were dissolved in 100 µl of 50 mM Tris-HCl
buffer (pH 8.5) containing 6 M guanidine HCl and 0.5 mM dithiothreitol and treated at 37 °C for 1 h with
2 mM iodoacetate. The reduced and carboxymethylated protein
was extensively dialyzed against 5 mM HCl. The precipitates formed during dialysis were collected by centrifugation, washed twice
with water, and suspended in 500 µl of 50 mM Tris-HCl
buffer (pH 7.4) containing 2.5 nmol of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Worthington) and subsequently digested at 37 °C for 12 h. The resultant peptide fragments were applied to a C18
column (4.6 × 150 mm; Tosoh, Tokyo, Japan) and eluted with 0.05%
trifluoroacetic acid for 10 min, followed by a linear gradient of
0-80% acetonitrile in 0.05% trifluoroacetic acid over 60 min at a
flow rate of 1.0 ml/min. The elution was monitored dually by absorbance
at 215 and 330 nm. The peptide, which had strong absorbance at 330 nm,
was further purified by high pressure liquid chromatography under
chromatographic conditions similar to those previously described,
except a directly linear gradient of 5-50% acetonitrile was employed.
The fractions which showed absorbance at 330 nm were pooled and
subjected to sequence analysis.
Generation of Metal-free Enzyme
Two hundred micromoles of recombinant low specificity
D-TA was dialyzed against 50 mM Tris-HCl buffer
(pH 7.4) containing 10 µM PLP and 10 mM EDTA
for 12 h at 4 °C. The enzyme solution was further dialyzed
twice against the same buffer without EDTA for 12 h at 4 °C to
remove EDTA.
Equilibrium Dialysis Study
Ten nanomoles of the metal-free enzyme was dialyzed twice
against 1 liter of 50 mM Tris-HCl buffer (pH 7.4)
containing 10 µM PLP and various concentrations of
MnCl2 (0-50 µM) for 12 h at 4 °C.
The metal concentrations, both inside and outside the dialysis bag,
were then determined as described below.
Atomic Absorption Measurement
The metal ion concentration was determined with a mode FLA-1000
atomic absorption spectrometer (Nippon Jarrell-Ash, Uji, Japan).
 |
RESULTS |
Cloning of the dtaAS Gene--
The primers used for cloning of the
dtaAS gene by PCR were based on the NH2-terminal
amino acid sequence of the purified low specificity D-TA
from Arthrobacter sp. strain DK-38. PCR with the primers and
Arthrobacter chromosomal DNA as the template yielded an
amplified band of 90 bp. Only this band was constantly amplified under
different PCR conditions. The amplified DNA was then cloned into pUC118
in E. coli. Nucleotide sequencing of the 90-bp fragment showed the presence of an open reading frame (ORF) continuing over the
entire sequence. The deduced amino acid sequence of the PCR fragment
was in perfect agreement with the NH2-terminal amino acid
sequence determined from the purified low specificity D-TA from Arthrobacter sp. strain DK-38 (16). We then directly
performed colony hybridization with the 90-bp fragment as a probe
against the established genomic library of Arthrobacter sp.
strain DK-38; three positive recombinant E. coli clones were
obtained from about 7,000 transformants. One of the clones showing
D-TA activity (0.1 units/mg) was chosen for further
characterization.
Sequence Analysis of the dtaAS Gene--
The plasmid, pUDTA,
extracted from the positive clone containing an approximately 7.2-kb
insert, was directly used as the template for sequencing the
dtaAS gene by the gene-walking method; the initial
sequencing primer was designed based on the nucleotide sequence of the
90-bp PCR product.
Sequence analysis of the double strand DNA revealed that the ORF
consists of 1,140 bp starting with an initiation codon, ATG, and ending
with a termination codon, TGA (Fig. 1). A
probable ribosome-binding sequence, GGAG, is present eight bases
upstream of the putative translational start codon (26). The
379-residue enzyme as deduced from the DNA sequence has a molecular
mass of 40,035 Da and composition as follows:
Ala55-Cys8-Asp25-Glu20-Phe10-Gly34-His10-Ile17-Lys7-Leu36-Met5-Asn9-Pro22-Gln20-Arg22-Ser20-Thr12-Val37-Trp3-Tyr7.
The NH2-terminal amino acid sequence coincided with
that of the purified enzyme determined by Edman degradation (Fig.
1).

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Fig. 1.
Nucleotide and deduced amino acid sequences
of the dtaAS gene and its flanking regions. The
asterisk denotes a translational stop codon. A putative
Shine-Dalgarno sequence is indicated as SD with a
double line. The thinly underlined amino acid
sequence is identical to that determined for the purified enzyme by
Edman degradation, and the boldly underlined amino acid
sequence is identical to that determined for the NaBH4
modified pryidoxyl peptide, except for Lys59 (see text for
details).
|
|
Sequence Homology with Other Proteins--
The predicted amino
acid sequence showed no significant similarity to those of the
currently known pyridoxal enzymes. However, in a search of protein
amino acid and nucleotide sequence data bases (GenBank, EMBL, DDBJ, and
Protein Data Bank) by means of the sequence similarity searching
programs Blast (19) and Fasta (20), D-serine deaminase
(GenBank, U41162) of Burkholderia cepacia and a hypothetical
protein (GenBank, U73935) of Shewanella sp. strain SCRC-2738
were found to be significantly similar in primary structure to low
specificity D-TA. It should be mentioned that the property
of the D-serine deaminase from B. cepacia was not reported, nor had this protein any similarity in primary structure with those of the extensively studied D-serine deaminase
from E. coli (27) and a probable D-serine
deaminase of Bacillus subtilis (GenBankTM,
D84432), although the E. coli and B. subtilis
proteins had as much as 55% identity and 69% similarity to each
other. The hypothetical protein of Shewanella is encoded by
an unnoted ORF, approximately 500 bp downstream of the eicosapentaenoic
acid synthesis gene cluster of Shewanella sp. strain
SCRC-2738 (28). The amino acid sequence alignment of
D-serine deaminase and the hypothetical protein with that
of low specificity D-TA is depicted in Fig. 2. D-Serine deaminase of
B. cepacia and the hypothetical protein of
Shewanella had 24 and 22% identities and 41 and 39%
similarities to those of low specificity D-TA,
respectively. Remarkably, Lys59 of low specificity
D-TA was completely conserved in the three proteins (Fig.
2).

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Fig. 2.
Sequence alignment of the low specificity
D-TA from Arthrobacter sp. strain DK-38 with
other proteins. From top to bottom in each
set, the proteins were the low specificity D-TA from
Arthrobacter sp. DK-38, D-serine deaminase
(GenBankTM U41162) of B. cepacia, and a
hypothetical protein (GenBankTM U73935) of
Shewanella sp. strain SCRC-2738. The similar amino acid
residues and identical residues in bold letters and
shading are boxed. The numbers on the
left and right are the residue numbers for each
amino acid sequence. The putative active site lysine residue has an
asterisk.
|
|
Overexpression of the dtaAS Gene in E. coli Cells--
The whole
dtaAS gene amplified by PCR directly from
Arthrobacter chromosomal DNA, with a putative Shine-Dalgarno
sequence (GGAG) and an initiation codon (ATG), was inserted into the
EcoRI site of pKK223-3. The resultant plasmid pKDTA was
introduced into E. coli XL1-Blue MRF' cells. The nucleotide
sequence of the whole amplified gene was further confirmed to have
undergone no error matching during the PCR by sequencing of the double
strands. The recombinant cells produced a large amount of low
specificity D-TA, and the specific activity of the crude
extract of E. coli XL1-Blue harboring pKDTA was elevated to
1.8 units/mg, which is about 180-fold over that of
Arthrobacter sp. DK-38 (see Ref. 16, Fig.
3, and Table
I). The protein was only produced in the
presence of IPTG (data not shown), indicating that the tac
promoter is essential for the overexpression.

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Fig. 3.
Purification of the low specificity
D-TA from Arthrobacter sp. strain DK-38.
The enzyme was loaded on a 10% SDS-polyacrylamide gel electrophoresis
and stained with Coomassie Blue after electrophoresis. Lane
1, cell extract (20 µg); lane 2, ammonium sulfate
fractionation (20 µg); lane 3, Butyl-Toyopearl (20 µg);
lane 4, DEAE-Toyopearl (7 µg); lane 5, Gigapite
(5 µg); lane 6, molecular mass standards. The
numbers on the left indicate the molecular masses
of the marker proteins.
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Table I
Purification of the recombinant low specificity D-TA
from Arthrobacter sp. strain DK 38
Threonine aldolase activity was determined with D-threonine
as a substrate, as described under "Experimental Procedures."
|
|
Enzyme Purification--
The recombinant low specificity
D-TA from Arthrobacter sp. strain DK-38 was
purified by ammonium sulfate fractionation, Butyl-Toyopearl, DEAE-Toyopearl, and Gigapite chromatography steps (Table I). About 100 mg of purified enzyme was obtained from 51 g wet cells. The
purified enzyme showed a single protein band on SDS-polyacrylamide gel
electrophoresis with a molecular mass of about 40 kDa (Fig. 3), the
same as the value calculated from the deduced amino acid sequence of
the enzyme.
PLP Requirement--
The recombinant enzyme exhibited absorption
maxima at 278 and 417 nm, with an
A278/A417 ratio of 4.8 (curve 1, Fig. 4). The solution of the pure enzyme was distinctly yellow. As has been demonstrated with other PLP-containing enzymes, the absorption peak
around 417 nm is characteristic of an azomethine linkage between the
coenzyme and a protein amino group. Reduction of the enzyme with sodium
borohydride by the method of Matsuo and Greenberg (29) resulted in a
loss of the enzyme activity, with a disappearance of the absorption
maximum at 417 nm and a concomitant increase in the
A330 (data not shown). The reduced enzyme was
catalytically inert, and the addition of PLP did not restore the enzyme
activity. This result suggests that sodium borohydride reduces the
aldimine linkage of the internal Schiff base. The holoenzyme was
converted to the apoenzyme (curve 3, Fig. 4) by treatment
with 1 mM hydroxylamine at 4 °C for 30 min and then
dialyzed against 20 mM potassium phosphate buffer (pH 7.4).
The constructed apoenzyme did not show D-TA activity. However, the activity was restored to 78% that of the native enzyme with a corresponding recovery of the A417
(curve 2, Fig. 4) on dialysis against 20 mM
potassium phosphate buffer (pH 7.4) supplemented with 10 µM PLP. In contrast, the two analogs of PLP, pyridoxal and pyridoxamine 5'-phosphate, neither restored the enzyme activity nor
the A417 (data not shown). Resolution of low
specificity D-TA was also carried out by treatment of the
enzyme with cysteine. As shown in Fig. 4, inset, cysteine
caused the disappearance of the 417-nm peak with a concomitant
appearance of a peak at 330 nm (curves 2-5, Fig. 4,
inset). The new absorbance peak at 330 nm disappeared on
subsequent dialysis. This result indicates that cysteine resolved the
enzyme by combining with the enzyme-bound PLP (417-nm peak) to form the
more stable thiazolidine compound (330-nm peak) (30). All of these
results show that PLP forms a Schiff base with a lysine residue of the
low specificity D-TA from Arthrobacter sp.
strain DK-38.

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Fig. 4.
Absorption spectra of the purified
recombinant low specificity D-TA. Curve 1,
holoenzyme; curve 2, reconstructed enzyme by dialysis of the
apoenzyme against 20 mM potassium phosphate buffer (pH 7.4)
supplemented with 10 µM PLP; curve 3,
apoenzyme constructed by hydroxylamine treatment. The spectra were
taken with 20 mM potassium phosphate buffer (pH 7.4) at
20 °C at a protein concentration of 1.1 mg/ml. Inset,
variation in the absorption spectrum of the recombinant low specificity
D-TA after the addition of cysteine. Curve 1,
holoenzyme (4.3 mg/ml); curves 2-6, the absorption spectra
of the enzyme (4.3 mg/ml) after addition of 100, 200, 300, 400, and 500 µM neutral cysteine, respectively. Each curve was taken
10 min after the addition of cysteine.
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To determine the bound PLP content of low specificity D-TA,
5 mg of the enzyme was extensively dialyzed against 20 mM
potassium phosphate buffer (pH 7.4) containing 10 µM PLP;
the PLP concentrations inside and outside the dialysis bag were
subsequently determined according to the method of Wada and Snell (25),
and the difference between the inside and outside was taken as the PLP
content of the enzyme. The PLP content of the enzyme was determined in
triplicate to be 0.85, 0.88, and 0.94 mol/mol of 40-kDa subunits,
respectively, suggesting that the enzyme has the capacity to bind 1 mol
of PLP as a cofactor per mol of 40-kDa subunits.
Identification of the PLP-binding Site--
The enzyme was treated
with NaBH4 as described under "Experimental
Procedures," and the isolation of the modified pyridoxyl peptide is
depicted in Fig. 5. The amino acid
sequence of the isolated peptide, which showed the absorbance peak at
330 nm, was determined by the Edman degradation procedure with a model PPSQ-10 protein sequencer. The 13 steps of degradation, except the 10th
step, gave an identical amino acid sequence
50HDVALRPHAKAHK62 of the amino acid sequence
deduced from the dtaAS gene (Fig. 1). The 10th step, which
did not show an identifiable peak on the sequencer, was presumably the
cofactor-binding lysine residue. Remarkably, this lysine residue is
completely conserved in the three proteins aligned as previously
mentioned (Fig. 2).

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Fig. 5.
Isolation of pyridoxyl peptide from
trypsin-digested low specificity D-TA by reversed phase
high pressure liquid chromatography. Elution of pyridoxyl peptide
(downward) by absorbance at 330 nm and that of other
peptides (upward) was by absorbance at 215 nm. The collected
fraction showing an absorbance peak at 330 nm was further purified by
repeated high pressure liquid chromatography and subjected to a protein
sequencer for the amino acid sequence analysis.
|
|
Metal Requirement for the Aldolase Activity--
To qualitatively
analyze the bound metal ions of the purified recombinant low
specificity D-TA from Arthrobacter sp. strain DK-38, gel filtration (HiLoad Superdex 200, Amersham Pharmacia Biotech,
Uppsala, Sweden) was employed to remove free metal ions attached to the
enzyme. The bound metal ions of the enzyme were subsequently analyzed
with a mode ICPS-1000III sequential plasma spectrometer (Shimadzu,
Kyoto, Japan); the enzyme so treated was determined to contain no
detectable divalent cations, such as Mn2+,
Mg2+, Co2+, Ni2+, Fe2+,
and Ca2+, which were previously shown to be activators of
the wild-type low specificity D-TA from
Arthrobacter sp. strain DK-38 (16). Kinetic analysis further
showed that the gel-filtrated enzyme had the same
Vmax and Km values toward
DL-threo- and DL-erythro-phenylserine as those of the
metal-free ones treated with EDTA, supporting the finding that the
purified enzyme contained no bound metal ions, at least no activating
divalent cations. Our previous work already demonstrated that the
Mn2+ ion stimulated the enzyme to give the highest aldolase
activity among all cations examined (16). Mn2+ ion was thus
selected as a target for the present study. Kinetic constants of the
metal-free enzyme toward various compounds were determined in the
presence or absence of Mn2+ ion, and the results are
comparatively given in Table II. The Km values of the enzyme toward all substrates
examined were independent of the presence or absence of
Mn2+ ion, suggesting that the metal ion may not be
involved in the substrate binding. In contrast, the
Vmax values of the enzyme were significantly
increased in the presence of Mn2+ toward all examined
compounds.
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Table II
Steady-state kinetic constants of the metal-free low specificity
D-TA from Arthrobacter sp. strain DK-38 determined with and
without MnCl2
The enzyme activities were determined spectrophotometrically as
described under "Experimental Procedures." The values of the
kinetic constants are given as mean ± standard deviation of three
independent determinations.
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Furthermore, experiments were carried out to correlate the enzyme
activity and amount of bound Mn2+ ions of the recombinant
low specificity D-TA. The metal-free enzyme was dialyzed
twice against 1,000 volumes of 50 mM Tris-HCl buffer (pH
7.4) containing 10 µM PLP and 100 µM
MnCl2 for 12 h. Following gel filtration to remove
free Mn2+ ions, the amount of bound Mn2+ ions
of the enzyme was determined by atomic absorption spectrometry to be
less than 0.01 mol/mol of subunit. As a consequence of the low affinity
of the aldolase for the Mn2+ ion, the stoichiometry of
Mn2+ ion binding was thus performed by equilibrium dialysis
study. As shown in Fig. 6, the maximum
number of bound Mn2+ ions was determined to be 0.92 mol/mol
of subunit, suggesting that the recombinant enzyme could bind 1 mol of
Mn2+ ion per mol of subunit. The maximal enzyme activity
was restored on the binding of approximately 1 mol eq of
Mn2+ (Fig. 6).

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Fig. 6.
Correlation of the bound Mn2+ ion
content and catalytic activity of the recombinant low specificity
D-TA. The enzyme was dialyzed twice against 50 mM Tris-HCl buffer (pH 7.4) containing 0-50
µM MnCl2. The bound Mn2+ ion
content, expressed in moles of Mn2+ ion per mol of subunit
( ), was calculated from the difference of the inside and outside of
the dialysis bag. Mn2+ ion concentrations were measured
with an atomic absorption spectrometer. The D-threonine
aldolase activity ( ) was determined following the standard assay
method, except the Mn2+ ion concentration was adjusted to
the amount outside the dialysis bag. Correlation of enzyme activity and
the bound Mn2+ ion content was replotted ( ).
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 |
DISCUSSION |
We have previously reported the occurrence, isolation, and
catalytic properties of a novel enzyme, D-threonine
aldolase, that catalyzes the interconversion of
D-threonine/D-allo-threonine and
glycine plus acetaldehyde from Arthrobacter sp. strain DK-38 (16). To study the structural and functional relationships of the
enzyme, we here cloned the dtaAS gene encoding the enzyme from the genomic DNA of Arthrobacter sp. strain DK-38 and
expressed the gene in E. coli cells. Our data showed that
the recombinant low specificity D-TA from
Arthrobacter sp. strain DK-38, with a unique primary
structure, is a novel metal-activated pyridoxal enzyme.
The recombinant low specificity D-TA from
Arthrobacter sp. strain DK-38 was concluded to be a
metal-activated enzyme, because of the following: 1) the purified
enzyme contained no detectable metal ions; 2) the enzyme activity was
stimulated by exogenous metal ions (Fig. 6, Table II, and Ref. 16); and
3) stoichiometric analysis revealed that the enzyme could bind 1 mol of
Mn2+/mol of subunit and the saturation of the metal-binding
showed the maximal aldolase activity (Fig. 6). We now have insufficient data to illustrate exactly the binding mode of the divalent cation to
the enzyme. However, according to the three general coordination schemes for the binding of enzyme, metal, and substrate, (i) substrate bridge complex (E-S-M), (ii) metal bridge complex
(E-M-S) or S-(E/M), and (iii) enzyme bridge
(M-E-S), where E, S, and M represent enzyme, substrate, and metal, respectively (31), low specificity
D-TA probably follows the mode of the enzyme bridge complex
because 1) metal ion binding was independent of the presence of the
substrate (Fig. 6), which excluded the model of substrate bridge
complex, and 2) the fact that the Km value of the
enzyme was almost the same in the presence or absence of the metal ion
(Table II) highlighted that metal bridge complex was not the case.
The results presented in this paper confirmed that low specificity
D-TA is a PLP-dependent enzyme, and
Lys59 was identified to be the PLP-binding site of the
enzyme by chemical modification with NaBH4 (Fig. 5). To our
knowledge, this is the first report dealing with the identification of
a divalent cation-activated pyridoxal enzyme, although some monovalent
cations have been shown to be the activators of other
PLP-dependent enzymes, such as 2,2-dialkylglycine decarboxylase, tryptophanase, and tyrosine phenol-lyase (32). The roles
of Na+ and K+ in PLP-dependent
enzyme catalysis were recently reviewed by Woehl and Dunn (32). On the
basis of the x-ray structural information of the metal sites for three
PLP-dependent enzymes and advances stemming from solution
spectroscopic and mechanistic studies, Woehl and Dunn proposed two
types of mechanisms to explain the effects of monovalent metal ions on
catalytic activity as follows: (i) mechanisms involving the metal ion
in a static structural role wherein binding activates the enzyme by
simply stabilizing the catalytically active conformation of the protein
and (ii) mechanisms where the metal ion plays a dynamic role in which
binding selectively assists one or more of the protein conformational transitions essential for complementarity between enzyme site and the
structure of an activated complex (32). It would be interesting to
reveal whether divalent cations play the similar role in the low
specificity D-TA catalysis.
PLP-dependent enzymes catalyze manifold reactions in the
metabolism of amino acids. On the basis of a computer analysis,
Alexander et al. (33) classified most of the known
PLP-dependent enzymes into
,
, and
families
correlating in most cases with their regio-specificity. The
enzymes, with a few exceptions, catalyze the transformation of amino
acids in which the covalency changes are limited to the
-carbon atom
that carries the amino group forming the aldimine linkage to the
coenzyme, such as serine hydroxymethyltransferase, 5-aminolevulinate
synthase, and 8-amino-7-oxononanoate synthase. The
and
enzymes
catalyze
-replacement or
-elimination and
-replacement or
-elimination reactions, respectively. We have recently cloned and
determined the primary structures of several L-type
threonine aldolases, L-allo-TA from A. jandaei DK-39 (15) and three low specificity L-TAs
from S. cerevisiae S288C (13), Pseudomonas sp.
strain NCIMB 10558 (14), and E. coli
GS245.2 These four
L-type TAs with a significant amino acid sequence identity
to one another showed no structural similarity to other PLP-dependent enzymes (14), although they belong
reaction-specifically to the
family. In a search of protein
sequence data bases (GenBankTM, EMBL, DDBJ, and PDB) using
either the total or partial sequence containing Lys59 as a
central amino acid as a probe, low specificity D-TA from Arthrobacter sp. strain DK-38 showed neither similarity in
primary structure to the members belonging to the
,
, and
families nor to the four L-type TAs, suggesting that the
low specificity D-TA probably represents another new family
of pyridoxal enzyme.
In summary, our findings reported here showed that low specificity
D-threonine aldolase from Arthrobacter sp.
strain DK-38, with a unique primary structure, is a novel
metal-activated pyridoxal enzyme, and Lys59 of the enzyme
was determined to be the pyridoxal binding site. To study further the
role of Lys59 and the activation mechanism of the divalent
cation, site-directed mutagenesis experiments and spectroscopic studies
are on the way.
 |
ACKNOWLEDGEMENTS |
We thank Professors Hideyuki Hayashi and
Hiroyuki Kagamiyama who kindly provided us with
DL-erythro-phenylserine.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research from the Ministry of Education, Science, Sports and Culture of Japan and for research for the future from the Japan Society
for the Promotion of Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB010956.
¶
To whom correspondence should be addressed: Laboratory
of Biocatalytic Chemistry, Biotechnology Research Center, Toyama
Prefectural University, Kurokawa 5180, Kosugi Machi, Toyama 939-0398, Japan. Tel.: 81-766-56-7500; Fax: 81-766-56-2498; E-mail:
ryu{at}pu-toyama.ac.jp.
1
The abbreviations used are: PLP, pyridoxal
5'-phosphate; TA, threonine aldolase; IPTG,
isopropyl-
-D-thiogalactoside; LB, Luria Bertani; bp,
base pair(s); kb, kilobase; PCR, polymerase chain reaction; ORF, open
reading frame.
2
J.-Q. Liu, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, and H. Yamada, submitted for publication.
 |
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