Download A novel zinc-dependent D-serine dehydratase

Biochem. J. (2008) 409, 399–406 (Printed in Great Britain)
399
doi:10.1042/BJ20070642
A novel zinc-dependent D-serine dehydratase from Saccharomyces
cerevisiae
Tomokazu ITO*, Hisashi HEMMI*, Kunishige KATAOKA†, Yukio MUKAI‡ and Tohru YOSHIMURA*1
*Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Aichi, Japan, †Division of Material Sciences,
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Ishikawa, Japan, and ‡Nagahama Institute of Bio-Science and Technology, Nagahama
526-0829, Shiga, Japan
YGL196W of Saccharomyces cerevisiae encodes a putative
protein that is unidentified but is predicted to have a motif similar
to that of the N-terminal domain of the bacterial alanine racemase.
In the present study we found that YGL196W encodes a novel
D-serine dehydratase, which belongs to a different protein family
from that of the known bacterial enzyme. The yeast D-serine
dehydratase purified from recombinant Escherichia coli cells
depends on pyridoxal 5 -phosphate and zinc, and catalyses the
conversion of D-serine into pyruvate and ammonia with the K m and
kcat values of 0.39 mM and 13.1 s−1 respectively. D-Threonine
and β-Cl-D-alanine also serve as substrates with catalytic
efficiencies which are approx. 3 and 2 % of D-serine respectively.
L-Serine, L-threonine and β-Cl-L-alanine are inert as substrates.
Atomic absorption analysis revealed that the enzyme contains one
INTRODUCTION
Several D-amino acids have been discovered in eukaryotes,
including mammals, and they are reported to have various
physiological functions. For example, D-aspartate has been found
in the mammalian central nervous system and endocrine tissues
and has been suggested to be involved in the regulation of hormone
release [1,2] and melatonin [3] and testosterone syntheses [4]. DSerine occurs primarily in the mammalian brain, with the highest
concentrations in the regions that are rich in NMDA (N-methyl-Daspartate) receptors, and modulates brain function as a coagonist
of the NMDA receptor [5,6]. D-Alanine has been found in the
anterior pituitary gland and pancreas [7], but its physiological role
is currently unclear. To understand the function of D-amino acids
in eukaryotes, we have been studying the roles of D-amino acids
and their metabolizing enzymes in two yeasts, Saccharomyces
cerevisiae and Schizosaccharomyces pombe, which are model
organisms of eukaryotic cells. S. pombe, which utilizes several Damino acids as a nitrogen source, contains enzymes acting on
D-amino acids such as alanine racemase [8], serine racemase (PDB
accession number 1V71) and D-amino acid oxidase [8]. On the
other hand, S. cerevisiae cannot utilize D-amino acids as a nitrogen
source [9], although D-amino acids are incorporated into the cells
via a general amino acid permease (Gap1p) [10]. Moreover Damino acids show toxicity and inhibit S. cerevisiae cell growth
[10,11]. In Escherichia coli cells, several D-amino acids have been
reported to serve as a substrate of aminoacyl tRNA synthases. The
formation of D-aminoacyl tRNA is suggested to lower the amount
zinc atom per enzyme monomer. The enzyme activities toward
D-serine and D-threonine were decreased by EDTA treatment and
recovered by the addition of Zn2+ . Little recovery was observed
with Mg2+ , Mn2+ , Ca2+ , Ni2+ , Cu2+ , K+ or Na+ . In contrast,
the activity towards β-Cl-D-alanine was retained after EDTA
treatment. These results suggest that zinc is involved in the
elimination of the hydroxy group of D-serine and D-threonine.
D-Serine dehydratase of S. cerevisiae is probably the first example
of a eukaryotic D-serine dehydratase and that of a specifically
zinc-dependent pyridoxal enzyme as well.
Key words: convergent evolution, D-serine dehydratase, pyridoxal
5 -phosphate, reaction mechanism, Saccharomyces cerevisiae,
zinc enzyme.
of normal L-aminoacyl tRNAs and cause a delay of cell growth
[12]. In S. cerevisiae cells, D-tyrosine was found to serve as a substrate of tyrosine tRNA synthase [13]. It is possible that the formation of D-aminoacyl tRNAs is one of the reasons for the toxicity of
D-amino acids to S. cerevisiae cells. S. cerevisiae possesses several
protective systems against D-amino acid toxicity, such as D-TyrtRNATyr deacylase catalysing the deacylation of D-Tyr-tRNATyr
[13] and D-amino acid N-acetyltransferase [14]. D-Amino acid Nacetyltransferase catalyses the acetyl group transfer from acetyl
CoA to an amino group of various D-amino acids [14]. The
resultant N-acetylated D-amino acids are excluded from the S.
cerevisiae cells more efficiently than free D-amino acids [11].
Heterologous expression of the D-amino acid-metabolizing
enzyme also protects the S. cerevisiae cells from D-amino acid
toxicity. For example, S. cerevisiae cell growth was inhibited by
D-alanine but was rescued by the expression of S. pombe alanine
racemase [8]. Alanine racemase depends on PLP (pyridoxal
5 -phosphate) and catalyses the interconversion between L- and
D-alanine. The enzyme occurs ubiquitously in eubacteria, and is
responsible for the formation of D-alanine, an essential
component of the peptidoglycan layer of bacterial cell walls.
Eukaryotes, except S. pombe, have no homologous genes with
those of the bacterial alanine racemase. S. pombe probably
obtained the alanine racemase gene through horizontal transfer
from proteobacteria [8]. S. cerevisiae does not contain alanine
racemase but has two genes, YBL036C and YGL196W, encoding
proteins with a motif common to alanine racemase. X-ray
crystallography of Ybl036cp revealed that the protein is bound
Abbreviations used: 2,4-DNP, 2,4-dinitrophenylhydrazine; DsdSC, D-serine dehydratase of Saccharomyces cerevisiae ; IPTG, isopropyl β-Dthiogalactoside; LB, Luria–Bertani; MBTH, 3-methyl-2-benzothiazolinone hydrazone; NMDA, N -methyl-D-aspartate; PLP, pyridoxal 5 -phosphate; TCA,
trichloroacetic acid; TFA, trifluoroacetic acid.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
400
T. Ito and others
with PLP and the whole structure closely resembles that of the
N-terminal domain of alanine racemase [15]. An unidentified
hypothetical protein, Ygl196wp, shows weak sequence similarity
to the bacterial alanine racemase and, on the Pfam database
(http://www.sanger.ac.uk/Software/Pfam/), is predicted to have a
common motif with the N-terminal domain of alanine racemase.
In the present study, we cloned and expressed the YGL196W
gene into E. coli cells and purified the gene products. We have
demonstrated that YGL196W of S. cerevisiae encodes a PLPdependent D-serine dehydratase. The enzyme, which we name
DsdSC (D-serine dehydratase of S. cerevisiae), is probably the
first example of a eukaryotic D-serine dehydratase and belongs
to a different protein family from that of the bacterial D-serine
dehydratase [16]. DsdSC is also unique in its zinc dependency.
EXPERIMENTAL
Materials
Amino acids and lactate dehydrogenase from rabbit muscle were
purchased from Sigma–Aldrich. Glutamate dehydrogenase from
bovine liver was from Wako Pure Chemicals. 2,4-DNP (2,4dinitrophenylhydrazine) was from Kanto Chemicals. Restriction
enzymes were from Takara. KOD-plus DNA polymerase was
from TOYOBO. Synthetic oligonucleotides were from Fasmac.
Histidine-binding resin was from Novagen (EMD Bioscience).
DEAE-TOYOPEARL 650M was obtained from Tosoh. All other
chemicals were of analytical grade.
Bacterial strain and culture conditions
S. cerevisiae BY4742 (MAT α, his3∆1, leu2∆0, lys2∆0,
ura3∆0) and BY4742∆ygl196w strains were obtained from
Invitrogen. S. cerevisiae BY4742 cells were grown at 30 ◦C in
YPD [1 % (w/v) yeast extract/2 % (w/v) peptone/2 % (w/v)
glucose] or SC medium [17]. E. coli XL1-Blue and BL21(DE3)
cells were used for the plasmid construction and gene expression
respectively. Recombinant E. coli cells were cultivated at 37 ◦C
in LB (Luria–Bertani) medium containing 50 µg/ml ampicillin.
Cloning of the YGL196W gene
The chromosomal DNA of S. cerevisiae BY4742 was isolated with
an ISOPLANT Kit (Wako Pure Chemicals), and digested
with HindIII. The resultant chromosomal DNA fragment was used
as the template for the amplification of YGL196W by PCR with
KOD-plus DNA polymerase, and the following oligonucleotides
as primers: DsdSCup, 5 -ATGCTCGAGGTTCTATCTCAATATAAAGGGTGCTCAG-3 (forward primer, the XhoI site is underlined) and DsdSCrv, 5 -ACCAAGCTTACCATTTCTGAAAAGGTAACCAAACATCG-3 (reverse primer, the HindIII site is
underlined). The amplified DNA fragment was digested with
XhoI and HindIII, separated by agarose gel electrophoresis, and
purified with a GeneClean II kit (Q-BIO gene). The amplified
DNA was then ligated into pET15b (Novagen) digested with
XhoI and HindIII. The resultant plasmid, pDsdSC, encodes the
Ygl196wp with an N-terminal tag, which consists of six histidine
residues and ten other amino acids residues providing a thrombin
cleavage site. Construction of the plasmid was verified by DNA
sequencing.
Expression and purification of the recombinant D-serine
dehydratase
The E. coli BL21(DE3) cells harbouring pDsdSC were grown
at 37 ◦C in LB medium containing 50 µg/ml ampicillin. When
D610 reached 0.5, YGL196W was expressed by the addition of
c The Authors Journal compilation c 2008 Biochemical Society
IPTG (isopropyl β-D-thiogalactoside) to a final concentration
of 0.5 mM, and the cultivation was continued at 30 ◦C for a
further 3 h. All subsequent steps were carried out at 0–4 ◦C. The
E. coli cells (wet weight of 12 g) were harvested by centrifugation
(4000 g for 5 min at 4 ◦C), resuspended in binding buffer
consisting of 20 mM Tris/HCl (pH 7.9), 500 mM NaCl and 5 mM
imidazole, and sonicated with a UP2005 Ultraschallprozessor
(Hielscher Ultrasonics). The lysate was centrifuged at 20 000 g
for 30 min, and the supernatant was applied to a Ni2+ -chelating
column (5 ml) pre-equilibrated with the binding buffer. The column was washed with 50 ml of washing buffer, consisting of
20 mM Tris/HCl (pH 7.9), 500 mM NaCl and 80 mM imidazole.
Ygl196wp was eluted with elution buffer consisting of 20 mM
Tris/HCl (pH 7.9), 500 mM NaCl and 1 M imidazole. The enzyme
solution was dialysed against PBS0 buffer (pH 7.5) consisting of
10 mM Na2 HPO4 , 2.7 mM KCl, 1.8 mM KH2 PO4 , 20 µM ZnCl2
and 20 µM PLP, and then applied to the DEAE-TOYOPEARL
column (7 ml) pre-equilibrated with PBS0 buffer. The column
was thoroughly washed with the PBS0 buffer containing 20 mM
NaCl, and the enzyme was eluted with a linear gradient of 20–
80 mM NaCl in PBS0 buffer. The fractions showing the enzymatic
activity were pooled and concentrated by ultrafiltration. Purity of
the enzyme was confirmed by SDS/PAGE.
UV-visible spectrum
The absorption spectrum of Ygl196wp was recorded in PBS
buffer (pH 7.5) consisting of 140 mM NaCl, 2.7 mM KCl, 10 mM
Na2 HPO4 , 1.8 mM KH2 PO4 and 20 µM PLP, with a Shimadzu
UV-2450 spectrophotometer.
Electrophoresis
SDS/PAGE was performed using a 10 % gel by the method of
Laemmli [17a]. After electrophoresis, the gel was stained with
0.1 % Coomassie Brilliant Blue R-250 and destained with 7 %
acetic acid containing 5 % ethanol.
HPLC analyses
HPLC analyses were carried out with a Shimadzu SCL-10A
system (Shimadzu) equipped with a Cosmosil 5C18 column
(4.6 mm ×150 mm; Nacalai Tesque) unless otherwise stated.
Enantioselective determination of amino acids with HPLC
We examined whether Ygl196wp catalysed the reactions of
pyridoxal enzymes, such as racemization, decarboxylation and
dehydratation, with various amino acids as substrates. Decreased
amounts and formation of the amino acids during the Ygl196wp
reaction were analysed by the enantioselective measurement of
amino acids with HPLC after the amino acids were derivatized
to fluorescent diastereomers [18]. The Ygl196wp reaction was
carried out in a 200 µl mixture containing 50 mM Hepes/NaOH
buffer (pH 8.0), 20 µM PLP, 10 mM amino acid and an appropriate amount of enzyme at 30 ◦C for 1 h. The reaction was stopped
by the addition of 200 µl of 10 % TCA (trichloroacetic acid) and
incubated at 4 ◦C for 15 min. After centrifugation of the mixture
at 19 000 g for 10 min, the supernatant was extracted three times
with water-saturated diethyl ether for the removal of TCA. Then,
amino acids were derivatized with Boc-L-Cys-OPA (where Boc
is t-butoxycarbonyl and OPA is o-phthaladehyde) as described
previously [18]. Separation of the derivatized amino acids with
HPLC was accomplished by a linear gradient of 0–60 % mobile
phase B (47 % acetonitrile in a 0.1 M acetate buffer, pH 6.0)
in mobile phase A (7 % acetonitrile in a 0.1 M acetate buffer,
pH 6.0) in 60 min at a flow rate of 0.8 ml/min at room temperature
Yeast D-serine dehydratase
(25 ◦C). Elution was monitored with an RF-10A fluorescence
detector (Shimadzu) with excitation and emission wavelengths of
344 and 443 nm respectively. The method of the enantioselective
determination of amino acids was also used for the analyses of
D-serine contamination in D-threonine and β-Cl-D-alanine.
Identification of oxo acid produced from D-serine
The oxo acid formed from D-serine through the Ygl196wp reaction was identified with HPLC after the product was derivatized
with MBTH (3-methyl-2-benzothiazolinone hydrazone) as described previously [19]. The reaction mixture (200 µl) containing
50 mM Hepes/NaOH buffer (pH 8.0), 20 µM PLP, 1 mM D-serine
and 2 µg of Ygl196wp was incubated at 30 ◦C for 1 h. A control
experiment was carried out with a similar reaction mixture except
that D-serine was replaced by pyruvate or hydroxypyruvate. The
reaction was stopped by the addition of 200 µl of 10 % TCA. After
the reaction mixture was diluted 10-fold, a 240 µl portion was
withdrawn and mixed with 10 µl of 1 mM 2-oxoglutarate as an
internal standard. After centrifugation of the mixture at 19 000 g
for 15 min, 100 µl of the supernatant was mixed with 200 µl of
1 M sodium acetate buffer (pH 5.0) and 80 µl of 0.1 % MBTH.
The mixture was incubated at 50 ◦C for 30 min, and a 20 µl
portion was subjected to HPLC. The derivatized oxo acids were
separated by a linear gradient of 0–100 % buffer B [90 % acetonitrile, 0.1 % TFA (trifluoroacetic acid)] in buffer A (20 %
acetonitrile, 0.1 % TFA) over 15 min at a flow rate of 1.2 ml/min
at room temperature. Elution was detected with a SDP-10A UVvisible detector (Shimadzu) at a wavelength of 350 nm.
Ammonia assay
The amount of ammonia formed from D-serine was assayed with
an indophenol reagent as described previously [20]. A reaction
mixture (200 µl) consisting of 50 mM potassium phosphate
(pH 8.0), 20 µM PLP, various concentrations of D-serine and 1 µg
of DsdSC was incubated at 30 ◦C for 60 min. An aliquot of the
reaction mixture (112 µl) was withdrawn, and mixed with 40 µl of
a phenol/nitroprusside solution and 48 µl of alkaline hypochlorite
solution. The mixture was incubated for 45 min at 30 ◦C. The
intensity of the developed blue colour was measured at 635 nm.
Enzyme assays
Ygl196wp (DsdSC) was routinely assayed by measuring the
amount of pyruvate formed with 2,4-DNP. The reaction mixture
(50 µl) containing 50 mM Hepes/NaOH buffer (pH 8.0), 20 µM
PLP, 10 mM D-serine and an appropriate amount of the enzyme
was incubated at 30 ◦C for 15 min. The reaction was stopped by the
addition of 50 µl of 0.05 % 2,4-DNP in 2 M HCl. The mixture was
incubated for 5 min at 30 ◦C, followed by the addition of 100 µl
of ethanol and 125 µl of 10 M NaOH. After incubation of the
mixture for 10 min at room temperature, the absorbance at 515 nm
was measured. For kinetic analyses with D-serine and β-Cl-Dalanine, the pyruvate formed was assayed spectrophotometrically
by following the oxidation of NADH in the coupling system with
rabbit lactate dehydrogenase. The reaction mixture contained
50 mM Hepes/NaOH buffer (pH 8.0), 20 µM PLP, various
concentrations of each substrate, 0.3 mM NADH, 10 units of
lactate dehydrogenase and 1.5 µg of DsdSC in a final volume
of 1 ml. The reaction was started by the addition of DsdSC, and
the decrease in absorbance at 340 nm was monitored at 30 ◦C.
For kinetic analyses with D-threonine, the ammonia formed
was assayed spectrophotometrically by following the oxidation
of NADH in the coupling system with bovine glutamate
401
dehydrogenase. The reaction mixture contained 50 mM Hepes/
NaOH buffer (pH 8.0), 20 µM PLP, various concentrations of Dthreonine, 0.3 mM NADH, 5 mM 2-oxoglutarate, 10 units of glutamate dehydrogenase and an appropriate amount of DsdSC in a
final volume of 1 ml. The reaction was started by the addition of
DsdSC, and the decrease in absorbance at 340 nm was monitored
at 30 ◦C.
Determination of proteins
The concentration of DsdSC was assayed with its molar
absorption coefficient at 280 nm, 47850 M−1 · cm−1 , which was
estimated from the tyrosine and tryptophan contents [21].
EDTA treatment of DsdSC
To obtain the metal-free enzyme, DsdSC was dialysed against
PBS buffer containing 5 mM EDTA (pH 7.3) at 4 ◦C for 16 h,
followed by dialysis against PBS buffer (pH 7.3) without EDTA
under the same conditions.
Atomic absorption measurement
The amount of zinc atom in DsdSC was determined by atomic
absorption spectroscopy on a Varian SpectrAA-50 spectrometer.
Gel-permeation chromatography
To obtain the molecular mass of the enzyme, the purified DsdSC
was subjected to gel-permeation chromatography with a TSK
G3000 SW column (7.8 mm × 300 mm) (Tosoh) equipped on
a Shimadzu SCL-10A HPLC system. DsdSC was eluted with
50 mM potassium phosphate buffer (pH 7.0) containing 300 mM
KCl at a flow rate of 1 ml/min at room temperature. Elution of the
protein was monitored by measuring the absorbance at 280 nm.
The column was calibrated with β-galactosidase (molecular mass,
464 kDa), glutamate dehydrogenase (246 kDa), BSA (69.2 kDa),
ovalbumin (42.8 kDa) and ribonuclease A (13.7 kDa).
CD measurement
To evaluate the secondary structure, CD spectra from 200 to
260 nm were measured with 5 µM enzyme at room temperature
with a Jasco J-720WI spectropolarimeter with a 0.1 cm light-path
cell. To assess the thermostability of DsdSC, the CD at 220 nm
was measured with increasing temperature from 5 ◦C to 75 ◦C at
a rate of 1 ◦C/min.
RESULTS
Expression and purification of the recombinant Ygl196wp in
E. coli cells
To study the properties of Ygl196wp, we cloned and expressed the
YGL196W gene in E. coli cells. The recombinant cells produced
Ygl196wp with six N-terminal histidine residues and a thrombin
cleavage site when IPTG was added to the culture. Ygl196wp
was purified from the recombinant E. coli cells by Ni2+ -chelating
and DEAE-TOYOPEARL column chromatography. The purified
enzyme showed a single protein band upon SDS/PAGE with
a molecular mass of 50 kDa (Figure 1A), which is compatible
with that calculated from its amino acid sequence (50059.2 Da).
The absorption spectrum of the purified Ygl196wp exhibited
absorption maxima at 278 and 417 nm, suggesting that Ygl196wp
contained PLP (Figure 1B). The 417 nm peak was decreased by
the addition of NaCNBH3 with a concomitant increase in the
absorption maximum at 320 nm. These are the typical phenomena
c The Authors Journal compilation c 2008 Biochemical Society
402
Figure 1
T. Ito and others
SDS/PAGE (A) and absorption spectra (B) of DsdSC
The UV-visible spectra of the purified DsdSC (solid line), and that incubated with 1 mM NaCNBH3 for 5 min at 20 ◦C (dotted line) were recorded. Other conditions are described in the text.
occurring upon the reduction of an aldimine linkage between
PLP and the ω-amino group of the lysine residue of protein.
Identification of Ygl196wp as a D-serine dehydratase
We examined whether Ygl196wp catalysed the reactions of
pyridoxal enzymes, such as racemization, decarboxylation and
dehydratation, with various D- and L-amino acids as substrates.
After the reaction, D- and L-amino acids, oxo acids and ammonia
in the reaction mixture were assayed by enantioselective HPLC
measurement, the 2,4-DNP method, and indophenol methods
respectively. We found that Ygl196wp converted D-serine into oxo
acid and ammonia. To identify the oxo acid produced, which was
expected to be pyruvate or hydroxypyruvate, the reaction product
was modified with MBTH and subjected to HPLC analysis
[19]. As shown in Figure 2, the oxo acid formed from D-serine
was identified as pyruvate, not hydroxypyruvate. Stoichiometric
analysis revealed that 1 mol of D-serine was converted into 1
mol of pyruvate and ammonia (see Supplementary Table S1
at http:// www.biochemj.org/bj/409/bj4090399add.htm). During
the reaction, no hydrogen peroxide was produced, which was
confirmed by the horseradish peroxidase reaction with TOOS [Nethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt
dihydrate] and 4-aminoantipyrine [22]. The results indicated that
Ygl196wp was not a D-amino acid oxidase. Neither NAD+ nor
NADP+ was required in the Ygl196wp reaction, which suggested
that Ygl196wp was not a dehydrogenase. Eukaryotic serine
racemase is known to catalyse the dehydration of D- and L-serine
[23], but Ygl196wp showed no reactivity toward L-serine and had
no racemase activity. On the basis of these results, we concluded
that Ygl196wp is a D-serine dehydratase. We thus named
Ygl196Wp DsdSC (D-serine dehydratase of S. cerevisiae). DsdSC
is probably the first example of a eukaryotic D-serine dehydratase.
Figure 2
reaction
Identification of the oxo acid formed from D-serine by the DsdSC
Elution profiles of the MBTH-derivatized pyruvate (A) and hydroxypyruvate (B) are shown as
controls. That of the MBTH-derivatized reaction product from D-serine is shown in (C). Peaks 1
and 1 , and 3 and 3 correspond to the hydroxypyruvate and pyruvate derivatives respectively.
Because E- and Z-isomers are produced from each carbonyl compound by the reaction with
MBTH, each derivative gives two peaks [19]. Peak 2 corresponds to the MBTH-derivatives of
2-oxoglutarate added as an internal standard (see the Materials and methods section).
Sequence analysis
A Blast search on the protein databases with DsdSC as a query
sequence gave several unknown proteins. Most of them were
putative proteins of yeasts and fungi. Supplementary Figure S1
(at http:// www.biochemj.org/bj/409/bj4090399add.htm) shows
c The Authors Journal compilation c 2008 Biochemical Society
the alignment of the amino acid sequences of some of these
proteins. DsdSC showed little overall sequence similarity with
any identified pyridoxal enzymes, but had some similar motifs
Yeast D-serine dehydratase
Table 1
Effects of various compounds on DsdSC activity
Table 2
The reaction mixture (50 µl) containing 0.5 mg/ml of DsdSC and 5 mM of each compound
in the PBS0 buffer was incubated at 20 ◦C for 15 min. A 5 µl portion of the mixture was
withdrawn, and the DsdSC activity was assayed using the 2,4-DNP method with or without PLP.
Values are means +
− S.D. (n = 3).
Activity (%)
Compound
With PLP
Without PLP
None
Sodium cyanoborohydride
N -Ethylmaleimide
Hydroxylamine
Phenylhydrazin
EDTA*
100.0
20.9 +
− 0.2
19.5 +
− 1.7
64.6 +
− 2.6
97.1 +
− 4.6
10.2 +
− 1.6
96.7 +
− 3.3
12.1 +
− 1.3
8.7 +
− 1.8
23.7 +
− 2.7
4.2 +
− 2.2
10.0 +
− 1.6
403
Kinetic parameters of DsdSC
The K m and k cat values for D-serine, D-threonine and β-Cl-D-alanine were obtained with the
untreated and EDTA-treated DsdSCs. The rates of the reactions with D-serine or β-Cl-D-alanine
were measured by the lactate dehydrogenase coupling method with 0.2, 0.5, 1.0, 2.0, 5.0 and
10 mM of each substrate. Those with D-threonine were obtained by the glutamate dehydrogenase
coupling method under the same conditions. The kinetic parameters were obtained according
to the double reciprocal plots of the initial velocity versus the substrate concentration, which
gave straight lines with a correlation coefficient value, r , of more than 0.99. Other experimental
conditions are described in the text. N.D., not detected.
Untreated
Substrates
D-Serine
K m (mM) k cat (s ) K m (mM) k cat (s ) K m (mM) k cat (s−1 )
0.39
0.13
β-Cl-D-alanine 1.45
D-Threonine
EDTA-treated+2.5 µM ZnCl2
EDTA-treated
−1
13.1
0.43
0.22
−1
0.19
N.D.
0.98
1.67
N.D.
0.48
0.48
0.17
2.45
12.5
0.34
0.24
*EDTA treatment was carried out as described in the text.
to D-threonine aldolase of Arthrobacter sp. strain DK-38 [24,25]
as shown in Supplementary Figure S1. The primary structure of
DsdSC was completely different from those of bacterial D-serine
dehydratase [16] and eukaryotic serine racemase [23] possessing
D-serine dehydratase activity. Both enzymes showed similarity
to L-threonine dehydratase in their primary structures [16,23]
and had two characteristic common motifs. One is the Ser-XaaLys-Ile-Arg-Gly sequence, of which lysine is the PLP-binding
site (see [16] for the bacterial D-serine dehydratase and PDB
accession number 1V7l for the S. pombe serine racemases).
Another conserved motif is the glycine-rich sequence, which
is considered to interact with the phosphate group of PLP
[26]. DsdSC has neither overall sequence homology with these
enzymes nor the two conserved sequences mentioned above.
These results suggest that DsdSC belongs to a protein family
that differs from that of the bacterial D-serine dehydratase and
the eukaryotic serine racemase. This is compatible with the
prediction that DsdSC has a motif such as that in the N-terminal
domain of the alanine racemase, which belongs to the fold-type
III groups of pyridoxal enzymes [27]. In contrast, bacterial
D-serine dehydratase belongs to the fold-type II group [27].
Cofactors essential for enzyme activity
Upon reduction with NaCNBH3 , DsdSC lost its activity,
which was not recovered by the addition of PLP (Table 1).
Hydroxylamine and phenylhydrazine (both 5 mM) lowered the
DsdSC activity to 23.7 and 4.2 % respectively, when the activity
was measured in the absence of PLP, and to 64.6 and 97.1 %
when it was measured in the presence of PLP (Table 1). These
results suggest that PLP serves as a cofactor of DsdSC.
Figure 3
DsdSC
Effect of zinc on the D-serine dehydratase activity of EDTA-treated
The reaction mixture (1 ml) contained 50 mM Hepes/NaOH buffer (pH 8.0), 20 µM PLP, 10 mM
D-serine, 0.3 mM NADH, 1.5 µg of DsdSC, 10 units of lactate dehydrogenase and various
concentration of ZnCl2 . The k cat values were obtained with the method described in Table 2.
The upper and lower dotted lines show the k cat values of the EDTA-untreated and EDTA-treated
DsdSC respectively. Other experimental conditions are described in the text.
was no contamination of D-serine in the D-threonine and β-ClD-alanine used in the present study. No reactions were observed
with L-serine, L-threonine or β-Cl-L-alanine. Neither the D- nor
the L-form of alanine, cysteine, tyrosine or tryptophan served as
a substrate of DsdSC. The kinetic parameters of DsdSC in the
D-serine dehydratase reaction were obtained by the coupling
method with lactate dehydrogenase. The apparent K m and kcat values for D-serine were 0.39 mM and 13.1 s−1 respectively (Table 2).
Metal requirement for the dehydratase activity
Effect of pH on enzyme activity
The effect of pH on the DsdSC activity was examined (see
Supplementary Figure S2 at http://www.biochemj.org/bj/409/
bj4090399add.htm). The maximum activity was obtained at
around pH 8.0. Tris/HCl buffer showed an inhibitory effect.
Substrate specificity and kinetic properties of DsdSC
The substrate specificity of the enzyme was examined. In addition
to D-serine, DsdSC showed little activity towards D-threonine
and β-Cl-D-alanine with 3.2 and 1.7 % efficiency (kcat ) of that
towards D-serine respectively (Table 2). We confirmed that there
DsdSC was inhibited by EDTA treatment (Table 1). To examine
the metal-dependency of the enzyme, we attempted to obtain the
metal-free enzyme by the dialysis of DsdSC against 5 mM
EDTA for 16 h, followed by dialysis against the buffer
without EDTA. The activity towards D-serine of the resultant
enzyme was about 5–15 % of that of the EDTA-untreated enzyme,
and was fully recovered by the addition of 2.5–5.0 µM Zn2+
(Figure 3). Over 10 µM, zinc showed inhibitory effects (Figure 3).
No activation of the EDTA-treated enzyme was obtained with
monovalent cations, such as K+ or Na+ , and divalent cations,
such as Mg2+ , Mn2+ , Ca2+ , Ni2+ or Cu2+ (results not shown).
We carried out atomic absorption measurements, and found that
the purified DsdSC contained 1.02 mol of zinc per mol of the
c The Authors Journal compilation c 2008 Biochemical Society
404
Figure 4
T. Ito and others
Gel-permeation chromatography of the purified DsdSC
Elution profile of DsdSC on a TSK G3000 SW column is shown. DsdSC was eluted at approx.
7.9 min after injection. The inset shows the calibration curve obtained with a set of molecular
mass standards.
DsdSC monomer (the zinc content is the average value from
the duplicated measurements). In contrast, the EDTA-treated
enzyme with 5.6 % of the kcat value of the untreated enzyme
contained 0.07 mol of zinc per mol of the monomer (7 % of the
zinc content of the untreated enzyme).
The activity toward D-threonine was also decreased below
the detection limit by EDTA-treatment and recovered by the
addition of Zn2+ (Table 2). On the other hand, α,β-elimination
of β-Cl-D-alanine was slightly increased rather than decreased
by EDTA treatment (Table 2). These results suggest that zinc
is involved in the elimination of the hydroxy group of D-serine
and D-threonine. CD spectra were nearly the same with the
EDTA-treated and untreated enzymes (see Supplementary Figure
S3 at http://www.biochemj.org/bj/409/bj4090399add.htm),
suggesting that the gross conformation was not affected by zincbinding.
Figure 5 Effect of D-serine on the cell growth between the mother strain
and the ∆ygl196w mutant of S. cerevisiae BY4742
The cell growth between the mother strain (closed symbols) and the ∆ygl196w mutant (open
symbols) of S. cerevisiae BY4742 was compared at 30 ◦C on SC medium containing 0.5 %
L-proline as a nitrogen source instead of ammonium sulfate, and 0 (square), 1 (circle) or 5 mM
(triangle) D-serine.
Effect of exogenous D-serine on the growth of the S. cerevisiae
∆ygl196w strain
Exogenous D-serine has been reported to delay S. cerevisiae
cell growth [10,11]. We studied the possibility that DsdSC
participates in D-serine detoxification in yeast cells. The growth
of S. cerevisiae BY4742 cells and that of BY4742∆ygl196w
cells were compared in a medium containing D-serine. In
the presence of 1 mM D-serine, the ∆ygl196w strain showed
slight growth retardation; in contrast the mother strain showed
a similar growth rate to that without D-serine (Figure 5).
These results suggest that YGL196W contributes to D-serine
detoxification. In the presence of 5 mM D-serine, the mother
strain also suffered from growth retardation. The detoxification
effect of DsdSC is limited, effectively below 5 mM D-serine.
DISCUSSION
Subunit structure of DsdSC
Gel-permeation chromatography of the purified DsdSC gave one
peak at approx. 7.9 min corresponding to the molecular mass of
118 kDa (Figure 4). Because the calculated molecular mass of the
DsdSC monomer is 50059.2 Da, DsdSC probably exists as a
dimer. The EDTA-treated enzyme and the enzyme from which
the N-terminal histidine-tag was removed by thrombin digestion,
also gave a similar elution pattern upon gel filtration (results not
shown). Zinc or a histidine-tag did not affect the subunit structure
of DsdSC.
Effect of EDTA treatment on the thermostability of DsdSC
Thermostability of DsdSC with or without EDTA treatment was
compared by measuring the temperature-dependent CD change at
220 nm. As shown in Supplementary Figure S4 (at http://www.
biochemj.org/bj/409/bj4090399add.htm), the CD at 220 nm of
the EDTA-treated and untreated enzymes started to decrease
at approx. 50 and 60 ◦C respectively. The EDTA treatment
decreased the thermostability of DsdSC.
c The Authors Journal compilation c 2008 Biochemical Society
In the present study we demonstrated that YGL196W of
S. cerevisiae encodes a PLP- and zinc-dependent D-serine
dehydratase and named it DsdSC. DsdSC is probably the first
eukaryotic D-serine dehydratase, and its primary structure has
no similarity to that of the bacterial enzyme. PLP-dependent
enzymes whose structures have been solved to date belong to one
of the five distinct fold types [27,28]. Each enzyme belonging
to a different fold-type has completely different folding and has
probably evolved from a different ancestor. DsdSC is predicted
to have a motif similar to that of the N-terminal domain of the
bacterial alanine racemase. If this is the case, DsdSC is classified
into the fold-type III group of pyridoxal enzymes. In contrast, the
bacterial D-serine dehydratase belongs to the fold-type II group
[27]. DsdSC and the bacterial D-serine dehydratase are examples
of convergent evolution, which is also exemplified by aspartate
aminotransferase and D-amino acid aminotransferase [27,28].
DsdSC shows no overall sequence similarities with the known
pyridoxal enzymes; however, we found that DsdSC had some
similarity in segments with D-threonine aldolase of Arthrobacter
sp. strain DK-38 [24,25] (see Supplementary Figure S1).
D-Threonine aldolase is a pyridoxal enzyme and depends on
divalent cations. Although the overall sequence similarity between
Yeast D-serine dehydratase
D-threonine aldolase and DsdSC is approx. 10 %, both enzymes
possess several common amino acid sequence segments. One
of them, RPHAK59 AHKC of D-threonine aldolase, is conserved
in DsdSC as RAHVK57 THKT (see Supplementary Figure S1).
The Lys59 of D-threonine aldolase is the active-site lysine bound
with PLP [25]. The Lys57 of DsdSC is also considered to be
the PLP-binding site because DsdSC lost its activity and ability
to form a Schiff base with PLP by the mutation of Lys57 to an
alanine residue (results not shown). Arthrobacter D-threonine
aldolase is predicted to have similar folding to that of the bacterial
alanine racemase [29], suggesting that D-threonine aldolase and
DsdSC belong to the same superfamily of PLP enzymes (foldtype III group). However, both enzymes differ from each other in
their metal dependency. D-Threonine aldolase completely lost its
activity as a consequence of EDTA treatment and recovered it by
the addition of divalent cations, such as Mn2+ , Mg2+ , Co2+ , Ni2+ ,
Fe2+ and Ca2+ . No activation was observed with Zn2+ [24,25]. In
contrast with the D-threonine aldolase, DsdSC was dominantly activated with Zn2+ (Figure 3). To the best of our knowledge, DsdSC
is the only PLP enzyme that is activated specifically with Zn2+ .
Besides DsdSC and the Arthrobacter D-threonine aldolase,
several PLP enzymes have been reported to be activated by metals.
Tyrosine phenol-lyase [30], tryptophanase [31], dialkylglycine
decarboxylase [32] and E. coli D-serine dehydratase [33] are
activated by monovalent cations, and the eukaryotic serine
racemase is activated by divalent cations [34]. Whoel and Dunn
[35] proposed two roles for the monovalent cations in the
enzyme catalysis, structural and dynamic roles. The structural role
indicates that the metal binding activates the enzyme by stabilizing
the catalytically active conformation of the enzyme. The dynamic
role means that metals are bound with the protein at the transition
state and lower the activation energy. The EDTA-treated DsdSC
lost the catalytic activity towards D-serine and D-threonine but
retained that towards β-Cl-D-alanine (Table 2). The dehydration of
D-serine and D-threonine, and the α,β-elimination of β-Cl-Dalanine are expected to proceed through common steps, α-proton
abstraction and elimination of the leaving group on the β-carbon
[36]. As a leaving group, chloride is more efficient than a hydroxy
group. Chloride can probably be eliminated without the help of
zinc from the deprotonated complex of β-Cl-D-alanine and PLP.
In contrast, zinc is required for lowering the activation energy for
the hydroxy group elimination from the D-serine and D-threonine
moiety of the intermediate. The D-serine dehydratase activity of
DsdSC was correlated with the zinc content of the enzyme, suggesting that zinc was indispensable for the reaction. However, we
cannot rule out the possibility that the absolutely zinc-free enzyme
still has a little activity. We are currently investigating the zincbinding site to construct the absolutely zinc-free enzyme by sitedirected mutagenesis. The results in the present study also demonstrated that the EDTA treatment lowered the thermostability of the
enzyme. Zinc also plays a role in stabilizing the enzyme structure
as well as activating catalysis. X-ray crystallography of the enzyme currently in progress will provide a clue to elucidate the detailed roles of zinc in DsdSC, a unique zinc-dependent pyridoxal
enzyme.
DsdSC gave a little contribution to the detoxification of
D-serine (Figure 5). D-Amino acid toxicity has been explained
by the formation of the D-aminoacyl tRNA and the resultant
decrease in the size of the tRNA pool [12]. It is possible
that DsdSC prevents the cells from forming D-serine-tRNA
by lowering the D-serine concentration in the cells. Analysis
of the genetic interaction network of the S. cerevisiae genes
([37] and http://www.yeastgenome.org/) suggests that DsdSC
interacts with 1,3-β-D-glucan synthase (Fks1p) and β-glucan
synthesis-associated protein (Kre6p), which are related to
405
cell-wall synthesis. In the vegetative cell wall of S. cerevisiae, the
serine content reaches approx. 17 % of the total amino acids [38].
If the serine residue is in the L-form, it is possible that DsdSC
prevents the contamination of D-serine to the S. cerevisiae cell
walls. On the other hand, Vorachek-Warren and McCusker [39]
reported that the heterologous expression of the E. coli D-serine
dehydratase gene (dsdA) conferred resistance to 19 mM
D-serine and the ability to use D-serine as a nitrogen source on
S. cerevisiae strain S019 cells. The authors have developed a
new method of yeast gene disruption with dsdA as a selection
marker. These experiments, as well as the results in the present
study (Figure 5), suggest that intrinsic DsdSC is not sufficient
to reduce the toxicity of the high concentration of D-serine.
Studies of the YGL196W gene expression should be important
for understanding the physiological roles of DsdSC and D-serine.
This work was supported in part by the Grant-in-Aid for Scientific Research 19380059 (to
T.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology.
REFERENCES
1 D’Aniello, A., Di Fiore, M. M., Fisher, G. H., Milone, A., Seleni, A., D’Aniello, S., Perna, A.
and Ingrosso, D. (2000) Occurrence of D-aspartic acid and N -methyl-D-aspartic acid in rat
neuroendocrine tissues and their role in the modulation of luteinizing hormone and
growth hormone release. FASEB J. 14, 699–714
2 Wolosker, H., D’Aniello, A. and Snyder, S. H. (2000) D-Aspartate disposition in neuronal
and endocrine tissues: ontogeny, biosynthesis and release. Neuroscience 100, 183–189
3 Ishio, S., Yamada, H., Hayashi, M., Yatsushiro, S., Noumi, T., Yamaguchi, A. and
Moriyama, Y. (1998) D-Aspartate modulates melatonin synthesis in rat pinealocytes.
Neurosci. Lett. 249, 143–146
4 Nagata, Y., Homma, H., Lee, J. A. and Imai, K. (1999) D-Aspartate stimulation of
testosterone synthesis in rat Leydig cells. FEBS Lett. 444, 160–164
5 Mothet, J. P., Parent, A. T., Wolosker, H., Brady, Jr, R. O., Linden, D. J., Ferris, C. D.,
Rogawski, M. A. and Snyder, S. H. (2000) D-Serine is an endogenous ligand for the
glycine site of the N -methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. U.S.A. 97,
4926–4931
6 Snyder, S. H. and Kim, P. M. (2000) D-Amino acids as putative neurotransmitters: focus
on D-serine. Neurochem. Res. 25, 553–560
7 Morikawa, A., Hamase, K. and Zaitsu, K. (2003) Determination of D-alanine in the rat
central nervous system and periphery using column-switching high-performance liquid
chromatography. Anal. Biochem. 312, 66–72
8 Uo, T., Yoshimura, T., Tanaka, N., Takegawa, K. and Esaki, N. (2001) Functional
characterization of alanine racemase from Schizosaccharomyces pombe : a eucaryotic
counterpart to bacterial alanine racemase. J. Bacteriol. 183, 2226–2233
9 La Rue, T. A. and Spencer, J. F. (1967) The utilization of D-amino acid by yeast.
Can. J. Microbiol. 13, 777–788
10 Rytka, J. (1975) Positive selection of general amino acid permease mutants in
Saccharomyces cerevisiae . J. Bacteriol. 121, 562–570
11 Yow, G. Y., Uo, T., Yoshimura, T. and Esaki, N. (2006) Physiological role of D-amino
acid-N-acetyltransferase of Saccharomyces cerevisiae : detoxification of D-amino acids.
Arch. Microbiol. 185, 39–46
12 Soutourina, O., Soutourina, J., Blanquet, S. and Plateau, P. (2004) Formation of
D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine towards Escherichia coli. J. Biol.
Chem. 279, 42560–42565
13 Soutourina, J., Blanquet, S. and Plateau, P. (2000) D-Tyrosyl-tRNA(Tyr) metabolism in
Saccharomyces cerevisiae . J. Biol. Chem. 275, 11626–11630
14 Yow, G. Y., Uo, T., Yoshimura, T. and Esaki, N. (2004) D-Amino acid N-acetyltransferase of
Saccharomyces cerevisiae : a close homologue of histone acetyltransferase Hpa2p acting
exclusively on free D-amino acids. Arch. Microbiol. 182, 396–403
15 Eswaramoorthy, S., Gerchman, S., Graziano, V., Kycia, H., Studier, F. W. and
Swaminathan, S. (2003) Structure of a yeast hypothetical protein selected by a structural
genomics approach. Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 127–135
16 Marceau, M., McFall, E., Lewis, S. D. and Shafer, J. A. (1988) D-Serine dehydratase from
Escherichia coli . DNA sequence and identification of catalytically inactive glycine to
aspartic acid variants. J. Biol. Chem. 263, 16926–16933
17 Sherman, F. (1991) Getting started with yeast. Methods Enzymol. 194, 3–21
17a Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680–685
c The Authors Journal compilation c 2008 Biochemical Society
406
T. Ito and others
18 Hashimoto, A., Nishikawa, T., Oka, T., Takahashi, K. and Hayashi, T. (1992) Determination
of free amino acid enantiomers in rat brain and serum by high-performance liquid
chromatography after derivatization with N- tert -butyloxycarbonyl-L-cystein and
o-phthaldialdehyde. J. Chromatogr. 582, 41–48
19 Tanaka, H., Yamamoto, A., Ishida, T. and Horiike, K. (2007) Simultaneous measurement of
D-serine dehydratase and D-amino acid oxidase activities by the detection of 2-oxo-acid
formation with reverse-phase high-performance liquid chromatography. Anal. Biochem.
362, 83–88
20 Weatherburn, M. W. (1967) Phenol-hypochlorite reaction for determination of ammonia.
Anal. Chem. 39, 971–974
21 Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y. and Kagamiyama, H. (1991)
Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed
reactions and thermodynamic aspects of its substrate specificity. Biochemistry 29,
5469–5476
22 Choi, S. Y., Esaki, N., Yoshimura, T. and Soda, K. (1992) Reaction mechanism of
glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.
J. Biochem. 112, 139–142
23 Foltyn, V. N., Bendikov, I., De Miranda, J., Panizzutti, R., Dumin, E., Shleper, M., Li, P.,
Toney, M. D., Kartvelishvily, E. and Wolosker, H. (2005) Serine racemase modulates
intracellular D-serine levels through an α,β-elimination activity. J. Biol. Chem. 280,
1754–1763
24 Kataoka, M., Ikemi, M., Morikawa, T., Miyoshi, T., Nishi, K., Wada, M., Yamada, H. and
Shimizu, S. (1997) Isolation and characterization of D-threonine aldolase, a
pyridoxal-5 -phosphate-dependent enzyme from Arthrobacter sp. DK-38. Eur. J.
Biochem. 248, 385–393
25 Liu, J. Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S. and Yamada, H. (1998) 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. J. Biol. Chem. 273, 16678–16685
26 Marceau, M., Lewis, S. D., Kojiro, C. L. and Shafer, J. A. (1990) Disruption of active site
interactions with pyridoxal 5 -phosphate and substrates by conservative replacements in
the glycine-rich loop of Escherichia coli D-serine dehydratase. J. Biol. Chem. 265,
20421–20429
27 Grishin, N., Phillips, M. A. and Goldsmith, E. J. (1995) Modeling of the spatial structure
of eukaryotic ornithine decarboxylases. Protein Sci. 4, 1291–1304
Received 15 May 2007/11 October 2007; accepted 16 October 2007
Published as BJ Immediate Publication 16 October 2007, doi:10.1042/BJ20070642
c The Authors Journal compilation c 2008 Biochemical Society
28 Soda, K., Yoshimura, T. and Esaki, N. (2001) Stereospecificity for the hydrogen transfer of
pyridoxal enzyme reactions. Chem. Rec. 1, 373–384
29 Paiardini, A., Contestabile, R., D’Aguanno, S., Pascarella, S. and Bossa, F. (2003)
Threonine aldolase and alanine racemase: novel examples of convergent evolution in the
superfamily of vitamin B6-dependent enzymes. Biochim. Biophys. Acta 1647, 214–219
30 Milic, D., Matkovic-Calogovic, D., Demidkina, T. V., Kulikova, V. V., Sinitzina, N. I. and
Antson, A. A. (2006) Structures of apo- and holo-tyrosine phenol-lyase reveal a
catalytically critical closed conformation and suggest a mechanism for activation by K+
ions. Biochemistry 45, 7544–7552
31 Isupov, M. N., Antson, A. A., Dodson, E. J., Dodson, G. G., Dementieva, I. S.,
Zakomirdina, L. N., Wilson, K. S., Dauter, Z., Lebedev, A. A. and Harutyunyan, E. H.
(1998) Crystal structure of tryptophanase. J. Mol. Biol. 276, 603–623
32 Toney, M. D., Hohenester, E., Cowan, S. W. and Jansonius, J. N. (1993) Dialkylglycine
decarboxylase structure: bifunctional active site and alkali metal sites. Science 261,
756–759
33 Kojiro, C. L., Marceau, M. and Shafer, J. A. (1989) Effect of potassium ion on the
phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5 -phosphate
cofactor of Escherichia coli D-serine dehydratase. Arch. Biochem. Biophys. 268,
67–73
34 De Miranda, J., Panizzutti, R., Foltyn, V. N. and Wolosker, H. (2002) Cofactors of serine
racemase that physiologically stimulate the synthesis of the N -methyl-D-aspartate
(NMDA) receptor coagonist D-serine. Proc. Natl. Acad. Sci. U.S.A. 99, 14542–14547
35 Woehl, E. U. and Dunn, M. F. (1995) The roles of Na+ and K+ in pyridoxal phosphate
enzyme catalysis. Coord. Chem. Rev. 144, 147–197
36 Eliot, A. C. and Kirsch, J. F. (2004) Pyridoxal phosphate enzymes: mechanistic, structural,
and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415
37 Tong, A. H., Lesage, G., Bader, G. D., Ding, H., Xu, H., Xin, X., Young, J., Berriz, G. F.,
Brost, R. L., Chang, M. et al. (2004) Global mapping of the yeast genetic interaction
network. Science 303, 808–813
38 Briza, P., Ellinger, A., Winkler, G. and Breitenbach, M. (1990) Characterization of a
DL-dityrosine-containing macromolecule from yeast ascospore walls. J. Biol. Chem. 265,
15118–15123
39 Vorachek-Warren, M. K. and McCusker, J. H. (2004) DsdA (D-serine deaminase): a new
heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae .
Yeast 21, 163–171