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INTRODUCTION |
Sphingolipids are ubiquitous membrane components having a backbone
structure called long-chain base
(LCB)1 which is
N-fatty acylated and linked to various polar head groups. In
eukaryotes, sphingolipids such as sphingosine, sphingosine 1-phosphate,
and ceramide are known to play important roles as second messengers in
various cellular events including proliferation, differentiation,
senescence, apoptosis, and immune response (1).
Serine palmitoyltransferase (SPT: EC 2.3.1.50) catalyzes the pyridoxal
5'-phosphate (PLP)-dependent condensation reaction of
L-serine with palmitoyl-coenzyme A (CoA) to generate
3-ketodihydrosphingosine (KDS). This reaction is the first committed
step in sphingolipid biosynthesis, utilizing substrates that are shared
by other metabolic pathways, and has an activity lower than those of
other enzymes involved in sphingolipid biosynthesis. Therefore, SPT is
thought to be a rate-determining enzyme in the sphingolipid synthetic pathway and, accordingly, a key enzyme regulating the cellular sphingolipid content (2). Eukaryotic SPTs have been known as membrane-bound proteins, enriched in the endoplasmic reticulum with their catalytic sites facing the cytosol (3). Genetic studies have
shown that two different genes, LCB1 and LCB2,
are required for SPT activity in the yeast Saccharomyces
cerevisiae (4
6). Subsequently, mammalian homologues of the
LCB genes from mouse, human, and Chinese hamster ovary cells
have also been reported (7
9). The biochemical studies using the
Chinese hamster ovary cell mutants demonstrated that both the LCB1 and
LCB2 proteins are subunits of SPT (10, 11). There is a high sequence
homology between LCB1 and LCB2, and they are classified as new members of the PLP-dependent
-oxamine synthase subfamily (6).
Based on the finding that LCB1 does not have a PLP-binding motif while LCB2 carries a lysine residue expected to form a Schiff base with PLP,
LCB1 and LCB2 have been speculated as a regulatory unit and a catalytic
unit, respectively (6, 10, 11). There is, however, no biochemical
explanation for the regulation mechanism of the SPT reaction at
present. The purification of the native form SPT from any eukaryotes
has not been successful because of the extremely low content and
instability of this enzyme. We, too, tried to construct the expression
system of the mouse SPT complex in Escherichia coli (12).
Affinity tagged forms of mouse LCB proteins lacking the membrane-anchor
regions were coexpressed in E. coli as partially soluble
proteins, but the purified SPT complex did not show enzymatic activity.
As the only achievement, Hanada et al. (11) obtained an
active SPT complex from the Chinese hamster ovary cell mutants expressing a hexahistidine-tagged LCB1 protein. However, it is very
difficult to obtain a sufficient amount of the active enzyme for
detailed enzymological analysis from such purification sources.
Although sphingolipids are not typical membrane constituents in
prokaryotes, there are some exceptions. In strict anaerobes such as the
genera Bacteroides, Porpthyromonas, and
Prevotella, high levels of sphingolipids were found; in some
species their contents in the total extractable lipid came to 70% (13,
14). It has been reported that Bacteroides melaninogenicus
contains a water-soluble SPT, but the purification of this enzyme was
not successful (15). The Gram-negative obligatory aerobic bacteria Sphingomonas and Sphingobacterium are the genera
whose lipid composition and structure of their sphingolipids have been
investigated most extensively (16, 17). In cells of Sphingomonas
paucimobilis, the lipopolysaccharide in their outer membrane is
completely substituted by sphingoglycolipid, and its proposed structure
is
1-O-D-glucuronosyl-2-N-2'-hydroxymyristoyldihydrosphingosine (glucuronosyl ceramide) (18, 19).
We found that cells of S. paucimobilis EY2395T
and Sphingobacterium spiritivorum EY3101T
contain significant SPT activity and report here the purification to
homogeneity and characterization of SPT from S. paucimobilis EY2395T. The primary structure of the enzyme has been
deduced from the cloned gene, and the SPT protein was successfully
overproduced in E. coli. The results show that SPT from
S. paucimobilis is a prototype of eukaryotic enzymes.
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EXPERIMENTAL PROCEDURES |
Chemicals--
L-Serine and other natural
L-amino acids were obtained from Nacalai Tesque (Kyoto,
Japan). Palmitoyl-CoA and lauroyl-CoA were from Funakoshi (Tokyo,
Japan). Myristoyl-CoA, n-heptadecanoyl-CoA, stearoyl-CoA,
arachidoyl-CoA, palmitoleoyl-CoA, oleoyl-CoA,
O-phospho-L-serine,
-methyl-D,L-serine, and L-serine
methylester were from Sigma. Serinol and serinamide were from Aldrich.
3-Hydroxypropionic acid was from Tokyo Kasei Kogyo (Tokyo, Japan).
4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF) was from Roche
Molecular Biochemicals. The low molecular weight gel filtration
calibration kit, gel filtration calibration kit, phenyl-Sepharose
CL-4B, PD-10, SuperoseTM 12, and MonoQTM HR
5/5 were from Amersham Pharmacia Biotec. DEAE-Toyopearl 650M and
Butyl-Toyopearl 650M were from Tosoh (Tokyo, Japan). The CHT-II Econo-PacTM cartridge was from Bio-Rad Laboratories.
Competent E. coli JM109 was purchased from Nippon Gene
(Tokyo, Japan). E. coli BL21(DE3) pLysS and plasmid pET21b
were from Novagen. A plasmid pUC118 was from TaKaRa (Kyoto, Japan). All
other chemicals were of the highest grade commercially available.
Bacterial Strains and Growth Conditions--
S.
paucimobilis EY2395T and S. spiritivorum
EY3101T were gifts from Dr. Eiko Yabuuchi, Aichi Medical
University, Aichi, Japan. Each strain was grown in 1 liter of LB medium
in 5-liter flasks at 30 °C and 90 rpm. Cells were harvested in the
late exponential growth phase (after 8
9 h) and stored at
20 °C
before use.
Assay of the Enzyme Activity--
In the purification steps, the
SPT activity was measured according to the methods of Williams et al.
(20) with minor modifications. The enzyme solution was incubated in 100 µl of a standard SPT reaction buffer (100 mM HEPES-NaOH
buffer (pH 7.5) containing 0.1 mM EDTA, 5 mM
dithiothreitol, 10 µM PLP, 0.4 mM
palmitoyl-CoA, and 2 mM L-[3H]Ser
(37 kBq/mmol)) at 37 °C for 10 min. The reaction was terminated by
addition of 100 µl of 2 N NH4OH. The lipids
were extracted with CHCl3/CH3OH (2:1 (v/v)),
and the radioactivity of the [3H]KDS that formed was
measured. The radioactivity extracted from enzyme-negative control was
regarded as a background. For steady-state kinetic analysis,
[14C]KDS was added to the extraction solvent as the
internal standard to estimate recoveries throughout the entire
procedure. [14C]KDS was enzymaticaly synthesized from
[14C]serine using purified SPT. For TLC analysis,
[14C]serine (10 kBq/mmol) or
[14C]palmitoyl-CoA (10 kBq/mmol) was used as the
substrate. The lipids were extracted and separated on TLC plates
(Silica Gel 60, Merck) with a solvent of chloroform, methanol, and 2 N NH4OH (40:10:1 (v/v)).
[14C]Dipalmitoylphosphatidylcholine was added to the
extraction solvent as the internal standard. Radioactive lipids on the
TLC plates were visualized and their relative radioactivity was
determined using a BAS2000 Image AnalyzerTM (Fujifilm,
Tokyo, Japan).
Purification of SPT--
All purification procedures were
performed at 4 °C. The buffer of 20 mM potassium
phosphate (pH 6.5) containing 0.1 mM EDTA, 5 mM
dithiothreitol, 0.1 mM AEBSF (protease inhibitor), and 20 µM PLP was used in all the following procedures except
for the fast protein liquid chromatography steps. The cells (30 g wet weight) were suspended in 300 ml of the buffer and disrupted sonically (Branson Sonic Power, Sonifier model 450) at 20 kHz for 9 min. The
intact cells and debris were removed by centrifugation (10,000 × g, 40 min). After the addition of
(NH4)2SO4 (final concentration, 30% saturation) and centrifugation (100,000 × g, 60 min),
the supernatant solution was applied to a phenyl-Sepharose CL-4B column (2.5 × 20 cm) equilibrated with the buffer containing 30% saturated (NH4)2SO4. The enzyme activity was
eluted with 1 liter of linearly decreasing
(NH4)2SO4 concentrations (30 to
0%). After the addition of
(NH4)2SO4 (final concentration,
30% saturation) again, the active fractions were applied to a
Butyl-Toyopearl 650M column (2.5 × 20 cm) equilibrated with the
buffer containing 30% saturated (NH4)2SO4. The enzyme activity was
eluted with 1 liter of linearly decreasing
(NH4)2SO4 concentrations (30 to
0%). The pooled active fractions were dialyzed against 2 liters of the
buffer. The dialysate was then applied to a DEAE-Toyopearl 650M column
(2.5 × 20 cm) equilibrated with the buffer. The enzymes were
eluted with 1 liter of linear gradient from 0 to 500 mM
NaCl. The pooled active fractions were concentrated and dialyzed
against 1 liter of 10 mM potassium phosphate buffer (pH
6.5) containing 0.1 mM EDTA, 5 mM
dithiothreitol, 0.1 mM AEBSF, and 20 µM PLP.
The dialysate was applied to a CHT-II column (1.35 × 3.5 cm),
which had been connected to a fast protein liquid chromatography
system, equilibrated with the same buffer. The enzyme was eluted with a
50-ml linear gradient from 10 to 200 mM potassium phosphate
(pH 6.5). The eluted enzyme was dialyzed against 1 liter of 50 mM Tris-HCl buffer (pH 7.5) and then applied to a
Mono-QTM HR column (0.55 × 5 cm) equilibrated with
the same buffer. The column was washed with 50 mM Tris-HCl
buffer (pH 7.5) containing 150 mM NaCl and eluted with a
25-ml linear gradient from 150 to 200 mM NaCl (pH 7.5). The
active fractions were combined, concentrated to 1 ml, filtered, and
stored at 4 °C. When 30 g of cells were used as the starting
material, 350 µg of a pure preparation of SPT was obtained.
Amino Acid Sequencing--
The purified enzyme (8.9 nmol) was
carboxymethylated, desalted, and digested at 37 °C for 30 min with
lysyl endopeptidase (33 pmol). The digested peptides were isolated by
reversed-phase high performance liquid chromatography on a Cosmosil
5C18 AR-II column (2.0 × 150 mm) with a linear gradient from 0 to
60% acetonitrile containing 0.05% trifluoroacetic acid. The amino
acid sequences were determined using a Hewlett Packard G1005A protein
sequencing system.
Isolation and Sequencing of Genomic DNA Clones Encoding
SPT--
Based on the amino acid sequences of the SPT peptides, we
synthesized degenerate oligonucleotides to obtain partial DNA fragments encoding the SPT gene by PCR against genomic DNA from S. paucimobilis. The oligonucleotides were
5'-GA(TC)GC(TCAG)CC(TCAG)GA(TC)AT(TCA)GCICC-3' and
5'-GC(TCAG)GT(TG)AA(TGA)AT(AG)TA(TCAG)GG-3' corresponding to
the amino acid sequences DAPDIAP and PYIFTA, respectively (Fig. 5,
dashed lines). The genomic DNA of S. paucimobilis
was prepared by a standard method (21). PCR was performed using LA
Taq DNA polymerase (TaKaRa, Kyoto, Japan) under the
following conditions: 30 cycles of 94 °C for 30 s, (40+
t) °C for 30 s, and 72 °C for 1 min, then
72 °C for 10 min, where t denotes that the annealing temperature was successively increased by 0.25 °C at each cycle. The
PCR product was directly cloned into a pCRII vector (Invitrogen, Netherlands) and sequenced by the "Dye-Terminator Cycle Sequencing" kit and an ABI 373A DNA sequencer (PerkinElmer Life Sciences). To
obtain the full-length SPT gene, a genomic DNA library (1 × 106 recombinants) was screened with the
32P-labeled PCR product (846 base pairs) as a probe. The
library was constructed as follows. A genomic DNA from S. paucimobilis was partially digested by Sau3AI,
2.5-3.5-kilobase fragments were agarose gel-purified and ligated into
BamHI-digested pUC118, and these constructs were used to
transform E. coli JM109. Labeling of the probe and detection
of hybridized fragments were performed using the BcaBESTTM
labeling kit (TaKaRa, Kyoto, Japan) and Quick-HybTM
hybridization solution (Stratagene), respectively. Twelve positive clones of the first screening were isolated. Restriction mapping and
partial sequencing revealed that all 12 clones were derived from the
same gene. The complete DNA sequence was determined for both strands of
the three longest clones.
Gel Filtration--
The enzymes were applied to a
SuperoseTM 12 and fractionated at a flow rate of 0.5 ml/min
with an fast protein liquid chromatography system. Bovine pancreas
ribonuclease A (Mr 13,700), bovine pancreas chymotrypsinogen A (Mr 25,000), hen egg
ovalbumin (Mr 43,000), bovine serum albumin
(Mr 67,000), rabbit muscle aldolase
(Mr 158,000), bovine liver catalase
(Mr 232,000), horse spleen ferritin
(Mr 440,000), bovine thyroid thyroglobulin
(Mr 669,000), E. coli aspartate aminotransferase (Mr 43,573 × 2), and
E. coli branched-chain amino acid aminotransferase
(Mr 33,962 × 6) were used as standard proteins.
Spectrophotometric Measurement--
The absorption spectra of
SPT were recorded with a Hitachi spectrophotometer U-3300 at 25 °C.
The buffer solution for the absorption measurements contained 50 mM HEPES-NaOH (pH 7.5) and 0.1 mM EDTA. SPT was
equilibrated with the above buffer by gel filtration using a PD-10
(Sephadex G-25) column (Amersham Pharmacia Biotech) prior to the measurement.
Other Methods--
Protein concentration during the purification
procedure was determined with a BCA protein assay kit (Pierce Chemical)
using bovine serum albumin as a standard. The protein concentration of
purified SPT was determined spectrophotometrically using a molar
extinction coefficient of 2.83 × 104
M
1 cm
1 at 280 nm for the PLP
form of the enzyme. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out with an SDS-Tris system using 10% polyacrylamide gel
according to the procedure described by Laemmli (22).
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RESULTS |
SPT Activity in S. paucimobilis and S. spiritivorum--
S. paucimobilis and S. spiritivorum contain a large amount of sphingolipids as their cell
components (16, 17), and thus sphingolipid biosynthetic enzymes have
been expected to exist in these bacteria. Cell-free extracts
(100,000 × g supernatants) prepared by sonication of
cells of these strains were examined for SPT activity by incubation
with [14C]serine and unlabeled palmitoyl-CoA. The lipids
were extracted and subjected to TLC analysis. Some radioactive products
were detected for each extract (Fig. 1).
One of these products was indistinguishable in migration from KDS
formed by mouse liver microsomes. The cell fractionation experiment
indicates that the SPTs of these bacteria are water-soluble enzymes
(Fig. 1). Because the extracts of S. paucimobilis showed
higher SPT activity than that of S. spiritivorum, S. paucimobilis was selected as the starting material for the
purification of SPT.

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Fig. 1.
Thin-layer chromatography of radiolabeled
products obtained by assays of SPT of mouse liver microsomes, S. paucimobilis, and S. spiritivorum. Lanes 1 and
4, mouse liver microsomes as a reference; lane 2,
crude extract after sonication of S. paucimobilis;
lane 3, the supernatant after centrifugation at 100,000 × g of S. paucimobilis crude extract; lane
5, crude extract after sonication of S. spiritivorum;
lane 6, the supernatant after centrifugation at 100,000 × g of S. spiritivorum crude extract.
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Purification of SPT from S. paucimobilis--
The enzyme was
purified to homogeneity by five steps of column chromatography. As
shown in Fig. 2, the purified SPT showed a single protein band with an apparent Mr of
about 50,000 on SDS-PAGE. Cell-free extracts of S. paucimobilis contain a large amount of yellow pigment, which was
very difficult to remove by the initial two hydrophobic column
chromatographies. Table I summarizes the purification of SPT from the S. paucimobilis extract. About
350 µg of the protein could be obtained from 6 liters of S. paucimobilis culture. The purification yield was reproducibly over
100% probably because some coexisting inhibitory materials were
removed as the purification proceeded. The purified enzyme could be
stored at 4 °C in sterile capped vials for up to 2 months in 20 mM Tris-HCl buffer (pH 7.5) without loss of activity.

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Fig. 2.
SDS-polyacrylamide gel electrophoresis at
various steps of SPT purification. Lane 1, crude
extract after sonication and centrifugation at 100,000 × g (20 µg of protein); lane 2, phenyl-Sepharose
CL-4B column (10 µg of protein); lane 3, Butyl-Toyopearl
column (5 µg of protein); lane 4, DEAE-Toyopearl column (5 µg of protein); lane 5, CHT-II (hydroxyapatite) column (1 µg of protein); lane 6, Mono-Q column (1 µg of protein).
The samples (10 µl) were analyzed by SDS-polyacrylamide gel
electrophoresis on a 10% gel and stained with the Coomassie Brilliant
Blue R-250. Prestained Protein Marker, Broad Range, from New England
Biolabs was used as the molecular mass standard.
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Physical Characterizations--
The Mr of
the purified enzyme was estimated to be about 90,000 by gel filtration.
Electrospray ionization mass spectrometry (ESI-MS) gave a signal at
m/z 44,916. These results show that the native SPT from
S. paucimobilis has a dimer structure composed of two
identical subunits. The purified SPT had an absorption spectrum with
two peaks at 338 and 426 nm other than the protein absorption peak at
278 nm in 50 mM HEPES-NaOH (pH 7.5) containing 0.1 mM EDTA (Fig. 3). These
absorption peaks are characteristic of PLP enzymes, which contain the
cofactor bound to the
-amino group of a lysine residue in the active
site (23). The addition of serine to the enzyme gave rise to an intense
absorption band at 426 nm and a less intense band at 338 nm, which
indicates that 338 nm absorption represents the active species and that
the external aldimine complex was formed.

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Fig. 3.
Absorption spectrum of purified SPT. The
conditions were as follows: 50 mM HEPES-NaOH buffer, 0.1 mM EDTA, pH 7.5, 25 °C, 0.35 mg/ml enzyme.
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Catalytic Properties and Substrate Specificity--
The time
course of KDS formation by the purified SPT was almost linear for at
least 20 min, and this activity was proportional to the amount of
purified enzyme up to 100 ng under the reaction conditions mentioned
above. The optimum pH for KDS formation was 7.5
8.0. SPT from S. paucimobilis was not inhibited by halide ions. No inhibition of
SPT activity was found with relatively high concentrations of
palmitoyl-CoA (up to 10 mM).
The substrate specificity is summarized in Tables
II and III. To assess the specificity for
amino acids, we determined the production of [14C]KDS
derivatives from [14C]palmitoyl-CoA with 20 natural amino
acids or various serine analogs. Purified SPT (50 ng) was incubated
with 0.8 mM [14C]palmitoyl-CoA and 4 mM various amino as the substrate. The activity was
detected only for serine among the natural amino acids examined (data
not shown). Serine methylester and O-phosphoserine were converted to KDS at lower rates (5 and 3% of the mean levels of KDS
formed with serine, respectively). However, at present, we cannot
preclude the possibility that serine derived from serine methylester by
hydrolysis was metabolized to KDS. We also examined the inhibition
effect of excess amounts of nonradioactive competitors on the
[3H]KDS production from 0.1 mM
L-[3H]serine (Table II).
[3H]KDS production was inhibited about 80% by 4 mM nonradioactive L-serine under this assay
condition. The inhibition of the [3H]KDS production by 4 mM each of other natural amino acids except for cysteine
was 40% or less. The effect of cysteine can be ascribed to the
thiazolidine formation of cysteine with PLP (23).
O-Phosphoserine was the most effective, with
-methyl-DL-serine, 3-hydroxypropionate, and serine
methyester following in this order. Seriamide and serinol were
essentially inert. Among various acyl-CoAs examined, palmitoyl-CoA was
the best substrate (Table III). The
unsaturated bond of the acyl chain of CoA analogs did not significantly
influence the SPT activity. The activity chain length profile showed a
bell-shaped pattern, which peaked around C16.
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Table II
Effects of various amino acids on the formation of
[3H]KDS from L-[3H]serine
Purified SPT (50 ng) was incubated with 0.1 mM
L-[3H]serine, 0.8 mM palmitoyl-CoA and 4 mM of each nonradioactive competitor indicated. The levels
of radioactivity of [3H]KDS that formed are shown as a
percentage of the mean level of [3H]KDS formed in the absence
of the competitors. The other natural amino acids were inert as
competitors. Each value varied ±10% between experiments.
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Table III
Acyl-CoA specificity of purified SPT
Purified SPT (50 ng) was used. The levels of KDS are shown as a
percentage of the mean level of KDS formed with palmitoyl-CoA. Each
value varied ±10% between experiments.
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Kinetic Parameters of Native SPT--
Steady-state analysis of the
purified SPT was carried out. The kinetic parameters for SPT were
determined with respect to serine and palmitoyl-CoA. As shown in Fig.
4, the experimental data were analyzed
according to the ordered Bi-Bi mechanism (24). The
Km values for serine and palmitoyl-CoA were 4.2 and 0.87 mM, respectively, and the kcat
value was 140 min
1 (Table
IV).

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Fig. 4.
Kinetic characterization of native SPT from
S. paucimobilis. The enzyme assay was performed
as described under "Experimental Procedures."
[3H]Serine as the substrate and [14C]KDS as
the internal standard were used. Panel A, the apparent rate
constants (kapp) for the KDS formation were
plotted as a function of palmitoyl-CoA concentration. Each solid
line represents the theoretical curve according to the initial
velocity equation on the ordered Bi-Bi mechanism using the kinetic
parameters summarized in Table IV. Panel B, determination of
K ,
K , and
kcat. Primary plot of
[palmitoyl-CoA]/kapp versus
[palmitoyl-CoA] at various serine concentrations. The secondary
substrate (serine) concentrations were 1 mM (closed
circle), 2 mM (open circle), 4 mM (closed square), 10 mM
(open square), 20 mM (closed
triangle), 50 mM (open triangle).
Inset, the secondary plot of [palmitoyl
CoA]/kapp ordinate intercept replot
versus 1/[Ser].
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Table IV
Kinetic parameters of purified SPT
The data for the native SPT are from Fig. 4. The data for the
recombinant SPT were obtained by the same assay.
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Cloning of the SPT Gene--
The nucleotide sequence of one of the
three clones sequenced, SPT1 (GenBankTM
accession number AB055142), is shown in Fig.
5. SPT1 contains a 1260-base
pair open reading frame (65% GC content) encoding a protein of 420 amino acid residues. The amino-terminal protein sequence of purified
SPT was Thr-Glu-Ala-Ala-Ala-, indicating that the first methionine of
purified SPT had been cleaved by processing. The deduced amino acid
sequence of SPT is also shown in Fig. 5. The molecular weight of 44,916 obtained by ESI-MS is in good agreement with the value of 44,910 calculated from the deduced amino acid sequence of SPT without the
first methionine within experimental error.

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Fig. 5.
Nucleotide and deduced amino acid sequences
of S. paucimobilis SPT gene. The deduced amino
acid sequence is given below the nucleotide sequence. The putative
Shine-Dalgarno (SD) sequence is indicated by the double
underline. The internal amino acid sequence of SPT determined by
Edman degradation is indicated by the underline. The
annealing sites of the oligonucleotides for the degenerate PCR cloning
are indicated by the dashed underline. An
asterisk marks the termination codon. An open
circle marks the lysine residue predicted to bind PLP.
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Sequence Comparisons--
The non-redundant data bases at the
National Center for Biotechnology Information were scanned for amino
acid sequences similar to the S. paucimobilis SPT sequence
using the BLAST algorithm (25). The predicted S. paucimobilis SPT protein is related to proteins grouped as the
-oxamine synthase family. This gene family includes eukaryotic SPT
subunits, 5-aminolevulinic acid synthase in heme biosynthesis,
8-amino-7-oxononanoate synthase (AONS) in biotin biosynthesis, and
2-amino-3-ketobutyrate-CoA ligase in the threonine utilization pathway,
all of which catalyze chemically similar reactions using the cofactor
PLP (26-30). The amino acid sequence alignment of S. paucimobilis SPT and mouse LCB1 and LCB2 proteins is shown in Fig.
6. Overall sequence homology is found between these proteins except for the NH2-terminal
transmembrane region of mouse LCB proteins. SPT has 27% identity and
48% similarity with mouse LCB1 and 31% identity and 49% similarity
with mouse LCB2. Fig. 7 shows the amino
acid sequence alignment of S. paucimobilis SPT and
three prokaryotic enzymes in the
-oxamine synthase family. There are
33% amino acid identity and 56% similarity between SPT and AONS of
Bacillus sphaerious, 33% identity and 54% similarity between SPT and 2- amino-3-ketobutyrate-CoA ligase of Bacillus subtilis, and 36% identity and 55% similarity between SPT and 5-aminolevulinic acid synthase of Agrobacterium
radiobacter.

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Fig. 6.
Comparison of SPT protein sequences. The
deduced amino acid sequence of SPT protein from S. paucimobilis SPT is compared with those of both LCB1 and LCB2
proteins from mouse. Alignment analysis was performed with GENETYX
(Software Development Co., Fukuoka, Japan). Residues conserved among
all proteins are fulltone-inverted and those conserved between the two
of these members are halftone-inverted. An asterisk marks
the lysine residue predicted to bind PLP. Triangles mark the
residues corresponding to identified residues in the active site of
AONS from E. coli (35).
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Fig. 7.
Comparison of protein sequences of four
PLP-dependent acyl-CoA transferases. The deduced amino
acid sequence of SPT protein from S. paucimobilis SPT is
compared with those of four of the PLP-dependent acyl-CoA
transferases. Alignment analysis was performed with GENETYX.
SPT, S. paucimobilis SPT; AONS,
Bacillus sphaericus 8-amino-7-oxononanoate synthase;
KBL, B. subtilis 2-amino-3-ketobutyrate CoA
ligase; ALAS, A. radiobacter 5-aminolevulinic
acid synthase. Residues conserved among all proteins are
fulltone-inverted and those conserved among the three of those proteins
are halftone-inverted. An asterisk marks the lysine residue
predicted to bind PLP. Triangles mark the residues
corresponding to identified residues in the active site of E. coli AONS.
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Expression of the SPT Gene in E. coli--
In order to construct
the expression system for S. paucimobilis SPT in E. coli, the internal NdeI restriction site
(334ATGCAT) of SPT1 was changed to ATGCAC
without changing the codons by site-directed mutagenesis, and the new
restriction sites, NdeI and HindIII, were
introduced to SPT1 at the translation initiation and
termination sites, respectively, by PCR. The modified SPT1 was ligated into a pET21b vector, and the recombinant plasmid was used
to transform E. coli BL21(DE3) pLysS cells. The SPT produced was functional, and the product amounted to about 10
20% of the total
protein in the crude extract of E. coli. Because of the overproduction of the protein, the expressed SPT would be partially in
the apo form, but it could be converted to the holoenzyme by addition
of PLP to the cell lysate. The recombinant enzyme was purified to
homogeneity using DEAE-Toyopearl, Butyl-Toyopearl, and hydroxyapatite
column chromatographies. The recombinant SPT provided a 50-kDa band on
SDS-PAGE (data not shown). The NH2-terminal sequence of the
purified enzyme, Thr-Glu-Ala-Ala-Ala-, agreed with that of the native
enzyme. Thus, the first methionine of the recombinant SPT was similarly
removed by processing. The purified SPT showed a peak with
m/z = 44,899 on ESI-MS, which is in agreement with the
native SPT within experimental error. The catalytic properties of the
recombinant SPT was the same as that of the native SPT; there is no
significant difference between the two enzymes in their steady-state
kinetic parameters (Table IV).
 |
DISCUSSION |
Because large scale cultivation of strict anaerobic bacteria is
difficult and the unsuccessful purification of the B. melaninogenicus SPT has already been reported (15), we searched
for aerobic bacteria containing sphingolipids and chose S. paucimobilis as an alternative purification source. S. paucimobilis contains sphingolipids which form more than 30% of
the total extractable lipid (16, 19). There is a report that
14C-labeled fatty acids or amino acids were incorporated
into the sphingolipids of S. paucimobilis (31). These
findings suggest the possibility for this bacterium to contain SPT.
The most important finding is that the S. paucimobilis SPT
is water-soluble and is a dimer composed of two identical subunits. All
the eukaryotic enzymes examined so far are heterodimers, and both of
the subunits are membrane-bound proteins. Membrane localization of
eukaryotic SPT complexes seems reasonable because the product of this
enzyme is a hydrophobic lipid incorporated into membranes. The
relationship between cellular localization and the mechanism of the
product release of bacterial SPT is the next subject of research. How
is the reaction product, KDS, transferred to the membrane? Does SPT
interact with membrane in vivo? Do other sphingolipid biosynthetic enzymes also exist as water-soluble forms in S. paucimobilis? As for the subunit composition, we can reasonably
consider that bacterial SPTs are homodimers. Judging from the high
sequence homology between LCB1 and LCB2, ancestral SPT would have been a homodimer, and the gene was duplicated at some point early in the
evolution of eukaryotes. The functional benefit for the
heterodimerization, or the role of the LCB1 subunit, however, remains unknown.
The purified enzyme showed an absorption spectrum characteristic of the
PLP enzyme. The ratio in the peak height of the PLP-derived absorption
bands (338 and 426 nm) to the protein-derived band (278 nm) indicates
that SPT binds two PLP molecules per dimer. S. paucimobilis
SPT is not inhibited by halide ions, although SPT activity of B. melaninogenicus has been reported to be significantly inhibited
(15). The inhibition by high concentrations of palmitoyl-CoA, which has
been observed for the eukaryotic enzymes, was not detected in S. paucimobilis SPT (Fig. 4) as well as in the B. melaninogenicus enzyme (11, 15, 32).
The substrate recognition of S. paucimobilis SPT was not so
strict, especially for the acyl-CoA substrate, compared with the eukaryotic enzymes (11, 32). This observation might reflect the
difference in the biological functions of sphingolipids between prokaryote and eukaryote. In eukaryotes, because sphingolipid metabolites take part in the intra- and intercellular signal
transduction pathways, it would be necessary to regulate strictly their
chemical structures and amounts by the synthetic enzymes. On the other hand, such physiological functions are not known for bacterial sphingolipids.
As shown in Table II, O-phosphoserine inhibited the
[3H]KDS formation from
L-[3H]serine as potently as the
L-serine. This is consistent with the finding that
O-phosphoserine was converted to a KDS derivative. Both
serinamide and serinol, derivatives in which the carboxyl group of
serine is modified, are not potent competitors of
[3H]serine in the above reaction. The inhibitory effects
of 3-hydroxypropionate and
-methyl-DL-serine agreed with
the belief that they can form complexes with SPT, which mimic the
Michaelis complex and the external aldimine, respectively. These
results indicate that the carboxyl group of serine is essential for the
recognition of the amino acid substrates by SPT.
Sequence comparison between the S. paucimobilis SPT and
other prokaryotic enzymes of the
-oxamine synthase family shows that conserved amino acids are distributed throughout the entire sequence (Fig. 7). In addition to Lys-267 that forms a Schiff base linkage with
PLP, catalytically important residues identified by x-ray crystallography on AONS from E. coli are completely
conserved at the corresponding positions in SPT, such as His-159,
Asp-231, and His-234 (Figs. 6 and 7, asterisk and
triangles) (33). These residues interact directly with PLP,
and His-159 and Asp-231 are also conserved in other
PLP-dependent enzymes such as aspartate aminotransferases
from various organisms.
We have succeeded in construction of the overproduction system of SPT
in E. coli. The growth rate of E. coli was not
inhibited even after the expression was induced, and the SPT
overproduced was catalytically active. Until now, it has been thought
that the toxicity of KDS, the reaction product of SPT, is one of the reasons why the expression system of SPT in E. coli cannot
be constructed. However, the present results imply that KDS is not toxic to the E. coli host, or it is rapidly metabolized. The
recombinant SPT was catalytically and spectrophotometrically
indistinguishable from the native protein. There has been little
success in the overproduction of the enzymes in the sphingolipid
biosynthesis pathway, and almost no detailed research exists concerning
the enzymatic characterization of these enzymes. This work permitted enough SPT for the three-dimensional structural analysis of this protein, which is essential for elucidation of the reaction mechanism of this enzyme. We are now attempting crystallization and x-ray diffraction studies of the S. paucimobilis SPT. Information
obtained from the Sphingomonas enzyme would provide us with
insight into the more complex eukaryotic homologue.