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INTRODUCTION |
Rat liver L-serine dehydratase
(SDH)1 (deaminase) (EC
4.2.1.13 or threonine dehydratase (deaminase), EC 4.2.1.16) catalyzes the pyridoxal phosphate (PLP)-dependent deamination of
L-serine (L-threonine) to yield pyruvate
(
-ketobutyrate). SDH plays an important role in gluconeogenesis
because the enzyme activity is remarkably induced by the consumption of
high-protein diets, starvation, and other treatments (see Ref. 1 for a
review). The purified enzyme is a dimer with a
Mr 34,200 subunit (2-4). PLP binds to Lys-40 to
form a Schiff base, and its encompassing amino acid sequence,
Ser-Xaa-Lys-Ile-Arg-Gly, is well conserved among SDHs from rat (5),
human (6), tomato (7), Escherichia coli (8, 9), yeast (10),
and so forth. Moreover, these enzymes have a glycine-rich sequence at a
region 100-130 amino acid residues downstream of the PLP binding lysyl
residue (9). The importance of this motif in the interaction with the
coenzyme was assessed by the finding that substitution of the glycine
residues with aspartic acid residues impairs PLP binding to E. coli D-serine deaminase (11, 12). These two conserved
sequences suggest that SDHs have evolved from a common ancestral
protein (9).
PLP catalyzes a variety of enzyme reactions such as transamination,
decarboxylation, isomerization, elimination, and so on (see Ref. 13 for
a review). Thus far, the crystal structures of more than 10 PLP enzymes
are available (14). These enzymes are mainly from bacterial sources,
with the exception of aspartate aminotransferases from chicken and pig
livers. On the other hand, PLP enzymes are classified into at least
three types of
,
, and
families on the basis of their primary
sequences (15). SDH, which catalyzes
,
elimination, belongs to
the
family, whereas tryptophanase (14) and tyrosine phenol-lyase
(16), although using similar catalyzing reaction mechanisms, are
affiliated with the
family (15). Thus, classification based on the
sequence alignment does not always conform to that based on the
reaction mechanism. For insight into this problem, we feel that it is
vital to accumulate information about the crystal structure of the
family members, which are known to include tryptophan synthase (17) and
E. coli threonine deaminase (18). A crucial step toward this
goal is to obtain pure enzyme for crystallization. Purification of SDH
from rat liver was extremely hard because of its relatively low
abundance and the sacrifice of numerous animals (19-23). In this work,
we have developed a bacterial expression and purification procedure and
characterized the recombinant enzyme. Along with crystal data on other
PLP enzymes, our key finding that SDH is specifically cleaved into two
fragments by various proteases that can independently refold after
denaturation strongly suggests that this enzyme is composed of at least
two folding domains.
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EXPERIMENTAL PROCEDURES |
Materials--
Male Wistar strain rats (200 g) and male Japanese
white rabbits (2.5 kg) were purchased from Sankyo Labo Service.
Biochemical reagents were commercially available and used without
further purification.
Plasmid Construction--
Plasmid pCWOri+ was described
previously (24). The initial portions of E. coli mRNAs
are usually rich in A and U, and the expression of foreign DNAs in the
bacterium is often facilitated by making the relevant regions rich in A
or T. Thus, we introduced three silent mutations in codons 2-5
(i.e. from the native sequence 5'-GCTGCCCAGGAG to the
mutated sequence 5'-GCTGCTCAAGAT
(the introduced changes are underlined)) via polymerase chain
reaction mutagenesis. Plasmid pCWOri+ has a NdeI restriction
site (CA|TATG; | indicates the NdeI cutting site)
coincident with the initiation ATG codon and multicloning sites of
XbaI, SalI, PstI, and
HindIII downstream of the NdeI site. A foreign
DNA is to be inserted between the NdeI site and any of the
cloning sites. SDH cDNA had no NdeI site coincident with
the initiation ATG codon and also had no usable restriction site in the
3' noncoding region. Therefore, a NdeI site and a
HindIII site were created by polymerase chain reaction mutagenesis. For this purpose, oligonucleotides
5'-TGGCCTGCTCAAGATTCCCTGCACGTG-3' (the underlined sequence
is part of the NdeI site) and
5'-GGATAAAGAAGCTTGGGCCACTGTC-3' (the underlined sequence is
the HindIII site) were synthesized as the 5' and 3' primers,
respectively. The latter sequence is derived from the native antisense
strand sequence corresponding to positions 35-59 downstream of the TGA
stop codon (5'-GACAGTGGCCCACCCTTCTTTATCC-3') (3). With these two
primers, the SDH cDNA containing the mutations was amplified by
polymerase chain reaction. The polymerase chain reaction product was
then digested with HindIII to produce a HindIII cut site at the 3' end. Before ligation of the DNA, pCWOri+ was digested with NdeI, and the NdeI site was filled
with the Klenow enzyme. This linearized plasmid was further digested
with HindIII to remove the original insert, and the
resulting plasmid was ligated to the modified SDH cDNA. This
construct was designated pCW-SDH.
Enzyme Assay--
SDH activity was determined by the
dinitrophenylhydrazine method (19). The complete reaction mixture
consisted of 50 mM borate-KOH (pH 8.3), 50 mM
serine, and 50 µM PLP/enzyme in 0.25 ml. The absorption
coefficient of hydrazone is 11.6 mM
1, as
determined with authentic sodium pyruvate. One unit of enzyme activity
is defined as the amount that catalyzes the formation of 1 µmol of
pyruvate per minute at 37 °C.
Purification of Recombinant SDH--
All operations were carried
out at a temperature of 0 °C to 4 °C unless otherwise stated.
E. coli carrying pCW-SDH were cultured in 2YT medium (25)
containing ampicillin (50 µg/ml) at 37 °C. IPTG was added to make
a final concentration of 0.5 mM when the cell turbidity
measured at 600 nm reached about 0.4, and the culture was continued for
an additional 14 h. The cells suspended in 20 ml of 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 5 mM 2-mercaptoethanol received 10 mg of lysozyme. They were
kept at 0 °C for 15 min, followed by
80 °C for 30 min. After
sonication at 200 watts for 1 min, the suspension was clarified by
centrifugation at 10,000 × g for 15 min. The
supernatant from 1 liter of cultured cells was then put on a DE-52
column (Whatman; diameter, 2 cm; height, 6 cm) prewashed with 10 mM Tris-HCl (pH 8.0). The column was washed with 100 ml of
50 mM Tris-HCl (pH 7.8), 1 mM EDTA, and 5 mM mercaptoethanol. Ammonium sulfate was added at 28 g/100
ml to the flow-through fraction. After 1 h, the precipitate was
collected by centrifugation at 12,000 × g for 30 min
and dissolved in 5 ml of 10 mM Tris-HCl (pH 7.5). After a
brief centrifugation, the clarified solution was applied to a column of
Sephacryl S-200 (Pharmacia; 3.2 × 97 cm) equilibrated with 10 mM potassium phosphate (pH 7.8, 1 mM EDTA, 50 µM PLP, and 5 mM 2-mercaptoethanol. Fractions
with a high specific activity were pooled and put on an AH-Sepharose column (Pharmacia; 2 × 7 cm) equilibrated with 10 mM
potassium phosphate (pH 7.8). SDH appeared as a single peak at about 40 mM potassium phosphate when eluted by a linear gradient of
100 ml each of 10 mM and 100 mM potassium
phosphate (pH 7.8) containing 0.1 mM EDTA and 1 mM dithiothreitol. The fractions judged pure by SDS-PAGE
were collected and concentrated by a Collodion bag. The enzyme was kept
in portions on ice or at
80 °C without loss of activity for at
least a month.
Isolation of Large and Small Fragments--
Trypsinolysis
(L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin; Worthington) was done in 0.1 M Tris-HCl (pH 8.0)
at the substrate:protease ratio of 1,000:1 (w/w) and a temperature of
25 °C. The reaction was terminated by adding a 5-fold molar excess
of leupeptin over trypsin. The digest was subjected to 13.5% SDS-PAGE,
and the gel was soaked in 1 M KCl. The visualized large and
small bands were cut out and electroeluted in TAE (40 mM
Tris acetate, 1 mM EDTA) buffer (25). Alternatively, the digest (5 mg) was brought to 6 M urea and size-fractionated
on a Sephadex G-75 column (1.8 × 98 cm) equilibrated with 10 mM potassium phosphate (pH 6.8), 0.1 mM EDTA, 1 mM dithiothreitol, and 6 M urea. Two-ml
portions were fractionated by monitoring the absorbance at 280 nm. The
fractions containing the large or small fragments were dialyzed against
10 mM potassium phosphate (pH 6.8), 0.1 mM
EDTA, and 1 mM dithiothreitol overnight and concentrated by a Collodion bag.
Amino Acid Sequencing--
The amino acid sequence of the
polypeptide (100 pmol) was determined by automated Edman degradation on
a Shimadzu PPSQ-10 gas-phase sequencer.
Spectral Analyses--
Absorption and CD spectra were measured
using a Hitachi 320 spectrophotometer and a Jasco J-500C
spectropolarimeter, respectively. The
-helix content was estimated
by the method of Chen and Yang (26).
Other Methods--
Protein was determined by the method of
Bradford (27) with bovine serum albumin as the standard. SDS-PAGE (28)
and immunoblotting (29) were performed as described previously. The
monospecific IgG to rat liver SDH was described previously (2) and was
used to identify recombinant SDH in Fig. 1. The antibody against the recombinant enzyme was raised in rabbits, further purified by recombinant SDH-coupled agarose gel chromatography as described previously (30), and used for the experiments shown in Figs. 3 and 6.
Densitometry was examined using NIH Image software.
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RESULTS |
Expression of SDH cDNA--
E. coli JM109 cells
transformed with the recombinant vector pCW-SDH or the control pCW with
no insert were cultured in the presence of IPTG. The IPTG-induced SDH
activity in a crude extract was found to be about 2 units/mg. The
specific activity of SDH purified from rat liver was in the range of
150-989 units/mg (19-23); thus, a 75- to 500-fold purification
appeared to be necessary for homogeneity. A plausible cause of the low
expression was the instability of the enzyme in this strain. We then
resorted to E. coli BL21, which lacks both
ATP-dependent Lon protease (31) and OmpT outer membrane
protease (32). As expected, the BL21 cell extract was found to have an
enzyme activity 5-fold higher than that of the JM109 extract. Fig.
1A shows SDS-PAGE of the extracts. The IPTG-treated JM109 cell extract had a
Mr 35,000 protein corresponding to the subunit
of rat liver SDH. This band was more intense in BL21 cells than in
JM109 cells (lanes 2 and 4). Immunoblot analysis
with the IgG to rat liver SDH indicated that JM109 contained not only
the Mr 35,000 band but a faint band of
Mr 24,000. This band became more evident in an
aged preparation (Fig. 1B, lane 4) or in a
preparation from JM109 cells grown at 31 °C (data not shown), but it
was not found in the BL21 extract. Thus, it is thought that the low
expression of the enzyme in JM109 cells results from extensive
proteolysis.

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Fig. 1.
Expression of SDH in E. coli
JM109 and BL21 cells. A, JM109 and BL21 cells were
transfected with pCW-SDH. These cells were cultured in the presence or
absence of IPTG (0.5 mM) for 14 h, and 20 µg of the
supernatants of the cell lysates were electrophoresed on a 12.5%
SDS-polyacrylamide gel. The gel was stained with Coomassie Blue.
Lane 1, JM109 ( IPTG); lane 2, JM109 (+IPTG);
lane 3, BL21 ( IPTG); lane 4, BL21 (+IPTG).
Numbers at the left margin indicate marker proteins (× 10 3). B, immunoblot analysis of E. coli JM109 and BL21 extracts. Ten µg of the extracts from JM109
and BL21 cells cultured with or without IPTG were electrophoresed as
described above, blotted onto a nitrocellulose membrane, and probed
with anti-rat liver SDH IgG (2). An ECLTM detection kit
from Amersham was used. Lane 1, JM109 with pCW ( IPTG);
lane 2, JM109 with pCW-SDH ( IPTG); lane 3,
JM109 with pCW-SDH (+IPTG); lane 4, a preparation that had
been obtained 1 day earlier and kept on ice; lane 5, BL21
with pCW ( IPTG); lane 6, BL21 with pCW-SDH ( IPTG);
lane 7, BL21 with pCW-SDH (+IPTG); lane 8, a
preparation that had been obtained 1 day earlier and kept on ice. Note
that SDH was considerably expressed in BL21 cells without IPTG
(lane 2 in A and lane 6 in
B).
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Purification and Some Properties--
SDH was purified from
E. coli BL21 by the conventional procedure as described
under "Experimental Procedures." Approximately 15 mg of pure enzyme
were obtained from a 1-liter culture. The enzyme activity emerged as a
single peak on AH-Sepharose or DEAE-cellulose column chromatography.
The chromatography deprived PLP of SDH, as found with a colorless
preparation. The addition of PLP to the apoenzyme allowed absorption
maxima to be restored at 330 and 415 nm, as reported previously for rat
liver enzyme (20). Amino acid analysis indicated that the N terminus of
the recombinant enzyme was alanine, whereas that of the liver enzyme
was acetylalanine (4, 5). Thus, in E. coli, the N-terminal
methionine residue is removed from the enzyme by posttranslational
modification, but acetylation of the new terminal residue does not
occur. The native and subunit molecular weights were about 66,000 by
gel filtration and 34,000 by SDS-PAGE, respectively, indicating that the recombinant enzyme is a dimer like the liver enzyme. The
Km values for serine and threonine of the
recombinant enzyme were 67 and 50 mM, respectively, which
were almost comparable to those reported for the liver enzyme (20). The
Vmax value for serine of the recombinant enzyme
was about 200 units/mg. This was almost comparable to the
Vmax of liver enzyme of 155 units/mg by Nakagawa and Kimura (20), 350 units/mg by Inoue et al. (21), and 278 units/mg by Simon et al. (22) but differed greatly from that by Leoncini et al. (989 units/mg; Ref. 23).
Susceptibility of SDH to Proteases--
Fig. 1 suggested that SDH
was vulnerable to attack by proteases. Then the purified enzyme (the
apoenzyme form) was examined for susceptibility to various proteases.
The reactions were done at the substrate:protease ratio of 1,000:1 to
30:1 by w/w. As shown in Fig. 2, trypsin
and lysyl endopeptidase produced two fragments with molecular weights
of 22,000 (large fragment) and 12,000 (small fragment).
Staphylococcus aureus V8 protease also brought two fragments
with molecular weights of 21,000 and 13,000. Subtilisin cleaved the
enzyme into two fragments comparable to the tryptic fragments in size.
However, the enzyme was virtually or considerably resistant to arginyl
endopeptidase, chymotrypsin, and thermolysis at the concentrations
examined. Similar results were obtained with the holoenzyme (data not
shown). These results suggest that PLP does not protect against
proteolysis.

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Fig. 2.
Protease susceptibility of SDH. Two µg
of SDH were reacted with various proteases at 25 °C for 15 min. The
ratio of substrate:protease was 1,000:1 to 30:1 by w/w as shown above
each lane. After reaction, the mixtures were boiled with the denaturing
SDS-PAGE buffer (27) and subjected to 13.5% SDS-PAGE. The gel was
stained with Coomassie Blue. Numbers at the left margin
indicate marker proteins (× 10 3).
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Kinetics of Limited Proteolysis--
To clarify the relationship
between proteolysis and inactivation, an apoenzyme was incubated with
trypsin as described above, and aliquots of the reaction mixture were
withdrawn over time. Trypsin abolished the enzyme activity following
pseudo-first order kinetics (Fig.
3A). SDS-PAGE showed the
appearance of Mr 22,000 and
Mr 12,000 bands with time (Fig. 3B,
left panel). There was a stoichiometry between the loss of
enzyme activity and the disappearance of the parent band by proteolysis
(Fig. 3B, right panel). The monospecific IgG to
the recombinant enzyme predominantly reacted with the
Mr 22,000 band (Fig. 3C).

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Fig. 3.
Effects of trypsin treatment on the catalytic
activity and molecular weight changes of SDH as revealed by SDS-PAGE
and immunoblotting. SDH was incubated with trypsin in 0.1 M Tris-HCl (pH 8.0) at the substrate:protease ratio of
1,000:1 (w/w) at 25 °C. Aliquots (2 µg) thereof were removed at
the times indicated and mixed with a 5-fold molar excess of leupeptin
over trypsin ( ). The control enzyme received no trypsin and was
incubated at 25 °C ( ). They were subjected to enzyme assay
(A), SDS-PAGE and Coomassie Blue staining (B),
and immunoblotting (C). In B, total areas of the
Mr 35,000 (undigested),
Mr 22,000, and Mr 12,000 bands among the lanes were almost equal (left panel) and
thus normalized as 100 in each lane. Right panel: ( ),
disappearance of the parent band; +, appearance of the
Mr 22,000 band; , appearance of the
Mr 12,000 band. For comparison, the profile of
the disappearance of SDH activity by trypsin digestion (A)
was depicted again ( ). In C, the monospecific IgG to
recombinant SDH was used.
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The Sites Digested by Trypsin and S. aureus V8 Protease--
Next
we determined the proteolytic site. After treatment with trypsin, the
digest was subjected to SDS-PAGE, and the large and small fragments
were recovered from the gel. Edman degradation demonstrated that the
first 10 amino acid sequences of the large and the small fragments were
consistent with the N-terminal sequence of the intact protein and the
amino acid sequence of a peptide from Ala-221 to Gln-230, respectively.
Likewise, the cleavage site by S. aureus V8 protease was
identified to be between Glu-206 and Gly-207.
Proteolysis Destabilizes PLP Binding--
In this section, we
studied the spectral change of a nicked enzyme. For this, the
recombinant enzyme (the holoenzyme form) was digested with trypsin
until an over 95% loss of the enzyme activity occurred and was then
dialyzed extensively. The treated enzyme exhibited about 50% reduced
absorptions at 330 and 415 nm and showed almost no difference in
absorption at 280 nm compared with the control enzyme (Fig.
4A). Likewise, the apoenzyme
was digested with trypsin, followed by an incubation with 200 µM PLP and dialysis. This enzyme also showed about 50%
decreased absorptions at 330 and 415 nm compared with the
PLP-reconstituted enzyme (data not shown). CD is a good tool to explore
the secondary structure of protein. It is known that PLP itself gives
no CD spectrum between 300 and 480 nm. When bound to the apoenzyme, PLP
could induce a CD spectrum with positive ellipticities at 330 and 415 nm (Fig. 4B). Interestingly, the holoenzyme previously
nicked with trypsin gave no appreciable signal in this region.
Similarly, the apoenzyme previously nicked and then reconstituted with
PLP did not display a positive ellipticity at either 330 or 415 nm
(data not shown). On the other hand, both the intact and nicked
holoenzymes exhibited similar CD spectra in the far-UV region; their
-helices were estimated to be 54% and 51%, respectively (Fig.
4C). These results show that proteolysis fairly impairs
coenzyme binding without accompanying a gross change in the secondary
structure. We further addressed the question of whether the two
fragments are separable under nondenaturing conditions. To test this,
the trypsin-treated enzyme was applied to a Sephadex G-75 column. The
nicked enzyme appeared at the position identical to that of the intact
enzyme as monitored with the absorption at 280 nm, suggesting that both fragments still associate with each other.

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Fig. 4.
UV absorption and circular dichroism spectra
of native and trypsin-treated SDH. SDH (holoenzyme) was digested
with trypsin until a >95% loss of enzyme activity and was dialyzed
against two changes of 1 liter of 10 mM potassium phosphate
(pH 7.8), 0.1 mM EDTA, and 0.1 mM
dithiothreitol at 0 °C over a period of 14 h. A,
absorption spectra of native (solid curve) and
trypsin-treated (dashed curve) SDH. The concentration of SDH
used was 1 mg/ml in each case. CD spectra were measured with a
0.5-mm-thick cell in the 280-480 nm region (B) and with a
0.2-mm-thick cell in the 200-280 nm region (C).
Thick, thin, and dotted curves represent the CD
spectra of native SDH, nicked SDH, and buffer solution, respectively.
The protein concentration used was 0.9 mg/ml in B and 0.2 mg/ml in C.
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Refolding of Large and Small Fragments--
Accumulating crystal
data have revealed that many PLP enzymes constitute two (or three)
folding domains (13). If SDH is made up of distinct folding domains,
these domains may be capable of refolding independently under
renaturing conditions after denaturation. Thus, a nicked enzyme was
denatured with 6 M urea and subjected to separation on a
Sephadex G-75 column containing 6 M urea. The isolated
fragments were confirmed by SDS-PAGE (Fig.
5A). Urea was removed by
extensive dialysis, and the CD spectra thereof were measured in the
far-UV region (Fig. 5B). As a reference, an uncut enzyme was
processed in a similar manner. The renatured protein had a specific
activity of 144 units/mg and 41%
-helix, corresponding to 72% and
76% of the specific activity and
-helix of the unprocessed enzyme,
respectively. Under these conditions, the large and small fragments
independently refolded, and the calculated
-helices were 14% and
37%, respectively (Fig. 5C). Although it is unknown at
present to what extent the
-helices are included in the large and
small segments of the SDH polypeptide, the
-helix of the refolded
large fragment seems to be less than that of the small fragment or the
native protein. These results do not exclude the possibility that the
large fragment could not refold more efficiently than the small
fragment.

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Fig. 5.
Separation of tryptic fragments by Sephadex
G-75 column chromatography containing 6 M urea and CD
spectra of renatured preparations. A, a holoenzyme was
digested with trypsin, denatured with 6 M urea, and applied
to a Sephadex G-75 column as described under "Experimental
Procedures" (top panel). The purity of the separated
fragments (peaks 1 and 2) was checked by 13.5%
SDS-PAGE and Coomassie Blue staining (bottom panel).
Lane 1, small fragment; lane 2, large fragment;
lane 3, uncut protein. B, the dialyzed large
fragment (0.17 mg/ml) and small fragment (0.28 mg/ml) were measured for
CD in the far-UV region in the same manner as described in the Fig. 4
legend. As a reference, a holoenzyme was denatured with 6 M
urea and renatured by dialysis in the same manner as the tryptic
fragments (uncut SDH).
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Liver Enzyme Is also Susceptible to Trypsin--
It has been
proposed that protein folding in eukaryotes is cotranslational, whereas
that in prokaryotes is posttranslational (33). It is possible that the
folding of the recombinant enzyme may be different from that of liver
enzyme or that the absence or presence of N-terminal blocking may be
responsible for the different folding pattern in the recombinant and
liver SDHs. To test this possibility, liver enzyme was partially
purified through ammonium sulfate fractionation and gel filtration
(Fig. 6A) and subjected to
limited proteolysis followed by immunoblot analysis. The preparation
that was not treated with trypsin exhibited two bands of
Mr 22,000, and Mr 24,000 other than an intact band (Fig. 6B, lane 4).
Because no protein inhibitor was included in the course of
purification, these subbands were considered to be degradation products. Trypsin treatment increased the Mr
22,000 band accompanying the disappearance of the parent band (Fig.
6B). The result suggests that the liver enzyme is also
susceptible to trypsin.

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Fig. 6.
Liver SDH is also susceptible to
trypsin. One µg of a purified recombinant enzyme and 20 µg of
a liver preparation (Sephacryl S-200 fraction) were digested with 1 and
40 ng of trypsin, respectively, at 25 °C for 10 or 20 min and
subjected to 13.5% SDS-PAGE followed by Coomassie Blue staining
(A) or immunoblotting with anti-recombinant SDH IgG
(B).
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DISCUSSION |
SDH is widely spread in nature, but its physicochemical properties
are considerably different from species to species. For example, rat
(20) and sheep liver enzyme (34) is a dimer with Michaelis-Menten
kinetics with respect to the substrate, whereas yeast and E. coli biosynthetic threonine dehydratase, the first enzyme in the
isoleucine synthesis pathway, is a tetramer and is feedback-inhibited
by isoleucine and heterotropically activated by valine (8). E. coli catabolic threonine dehydratase induced anaerobically in
tryptone-yeast extract medium is a tetramer and is allosterically
activated by AMP (9). However, E. coli D-SDH is
a monomer (35, 36). (An appreciable amount of D-serine is
present in mammalian brain, which is produced by the racemization of
L-serine. The occurrence of D-SDH remains to be
determined (37)). The importance of the glycine-rich sequence in SDH
was first verified with D-SDH (11, 12), and its preliminary
crystal data were reported (38). Recently,
non-PLP-dependent, sulfur/iron-dependent SDH
was found in some microorganisms (39). It is obvious that this new type
of enzyme has no sequence homology to authentic SDH including the
sequence around the PLP-binding lysyl residue and the glycine-rich
motif (39), but it is unclear whether such an enzyme occurs in eukaryotes.
We have previously succeeded in isolating more than 80 mg of glycine
methyltransferase, a rat liver enzyme, from a 1-liter culture with a
combination of the pCW vector and E. coli JM109 (40), and
this led to crystallographic results (41). However, the same vector did
not work effectively on SDH. This was due largely, if not entirely, to
the high sensitivity of recombinant enzyme to endogenous protease(s).
The E. coli BL21 strain circumvented this problem. Even in
the absence of IPTG, a significant amount of SDH was seen in this
strain relative to JM109 (Fig. 1). This finding gave us a chance to
analyze a higher order structure of SDH by limited proteolysis.
S. aureus V8 protease and trypsin nicked at Glu-206 and
Lys-220 of the purified enzyme, respectively. Although the cutting
sites by subtilisin and lysyl endopeptidase remain undetermined, they
are expected to be near Lys-220, because similar fragments were
liberated by these proteases (Fig. 2). Moreover, a fragment appearing
in the JM109 cell extract was about Mr 1,500 larger than the tryptic large fragment, indicating that the cleavage
site would be around Leu-234. PLP did not protect against proteolysis,
suggesting that a stretch of about 30 residues susceptible to protease
does not lie in the PLP binding site but rather is exposed to a solvent phase.
It is said that proteins that consist of two independently folding
domains connected by a hinge peptide are particularly susceptible to
proteolysis (e.g. see Ref. 42 for a review). It is tempting to speculate that the protease-sensitive region of SDH acts as a hinge
for the two domains. For substantiating this notion, two tryptic
fragments were separated under denaturing conditions and dialyzed
extensively. CD measurements revealed that the two fragments could
spontaneously refold (Fig. 5B). Thus, these results support the notion that SDH is composed of at least two domains. In fact, most
PLP enzymes are known to have multiple folding domains; the two-domain
structure is found in aspartate aminotransferase (43), tryptophanase
(14), tyrosine phenol-lyase (16), dialkylglycine decarboxylase (44),
and tryptophan synthase (17), whereas the three-domain structure is
seen in glutamate-1-semialdehyde aminomutase (45) and cystathionine
-lyase (46).
Fig. 7 compares the diagrammatic
structure of SDH with those of the
subunit of tryptophan synthase
(17) and the biosynthetic threonine deaminase of E. coli
(18). SDH is considered to be close to the tryptophan synthase
subunit because (i) in both enzymes, the PLP binding lysyl residue is
located relatively early in the primary sequences and the distance
between this residue and the glycine-rich sequence is about 130 residues, and (ii) the reaction mechanisms of
replacement and
elimination are relatively similar. Tryptophan synthase
subunit was
nicked at Lys-272, Arg-275, and Lys-283 by various proteases to result
in the large and small fragments, and the isolated fragments could spontaneously refold under renaturing conditions (47). The x-ray crystallography revealed a 54-residue stretch containing the residues susceptible to proteases located at the N-terminal side of the C-domain
(17). Threonine deaminase was found to have the N-terminal catalytic
and C-terminal regulatory domains that are connected with a neck region
consisting of 12 amino acid residues (18). The folding pattern of the
N-domain resembles that of the tryptophan synthase
subunit, but the
structure of the regulatory domain is rather similar to that of the
regulatory serine binding domain in allosteric 3-phosphoglycerate
dehydrogenase (48). It remains unclear whether the neck region is
susceptible to proteases like SDH and tryptophan synthase
subunit.
At any rate, these similarities are supporting evidence that SDH is
comprised of two domains. Although large differences exist in
structural and kinetic properties between E. coli threonine
deaminase and rat liver SDH, the first indication of the x-ray
structure of the former among various hydroxyamino acid dehydratases
would be informative for the future study of rat liver SDH.

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Fig. 7.
Schematic representation of the structures of
the Salmonella typhimurium tryptophan synthase subunit,
rat liver SDH, and E. coli threonine deaminase. The
structures of S. typhimurium tryptophan synthase subunit
and E. coli threonine deaminase are taken from Refs. 17 and
18. Arrows indicate the location of the lysyl residue
capable of forming a Schiff base with PLP and the glycine-rich sequence
that interacts with the phosphate group of PLP. , the region
susceptible to proteases. The in threonine deaminase represents the
"Neck" according to the nomenclature of the authors.
, the C-domains assigned by x-ray analysis. A portion of the
N-terminal region contributes to the structure of the C-domain of
tryptophan synthase subunit. The structure of the C-domain of
threonine deaminase resembles that of the serine binding domain of
3-phosphoglycerate dehydrogenase.
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