From the Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
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ABSTRACT |
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Selenocysteine lyase is a pyridoxal 5'-phosphate
(PLP)-dependent enzyme that catalyzes the exclusive
decomposition of L-selenocysteine to
L-alanine and elemental selenium. An open reading frame,
named csdB, from Escherichia coli encodes a
putative protein that is similar to selenocysteine lyase of pig liver
and cysteine desulfurase (NifS) of Azotobacter vinelandii.
In this study, the csdB gene was cloned and expressed in
E. coli cells. The gene product was a homodimer with the
subunit Mr of 44,439, contained 1 mol of PLP as
a cofactor per mol of subunit, and catalyzed the release of Se,
SO2, and S from L-selenocysteine,
L-cysteine sulfinic acid, and L-cysteine,
respectively, to yield L-alanine; the reactivity of the
substrates decreased in this order. Although the enzyme was not
specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme
can be regarded as an E. coli counterpart of mammalian
selenocysteine lyase. We crystallized CsdB, the csdB gene
product, by the hanging drop vapor diffusion method. The crystals were
of suitable quality for x-ray crystallography and belonged to the
tetragonal space group P43212 with
unit cell dimensions of a = b = 128.1 Å and c = 137.0 Å. Consideration of the Matthews
parameter Vm (3.19 Å3/Da) accounts
for the presence of a single dimer in the crystallographic asymmetric
unit. A native diffraction dataset up to 2.8 Å resolution was
collected. This is the first crystallographic analysis of a protein of
NifS/selenocysteine lyase family.
Selenocysteine lyase
(SCL)1 (EC 4.4.1.16) and
cysteine desulfurase (commonly referred to as NifS) are pyridoxal
5'-phosphate (PLP)-dependent enzymes that catalyze the same
type of reaction, i.e. the removal of a sulfur or selenium
atom from L-cysteine or L-selenocysteine to
produce L-alanine. NifS acts on both L-cysteine and L-selenocysteine (1, 2). Several enzymes participating in sulfur metabolism also act on the selenium analogs of the substrates (3). In contrast, SCL exclusively decomposes
L-selenocysteine. Selenium is specifically metabolized by
such enzymes as selenophosphate synthetase (4), selenocysteine synthase
(5), and selenocysteine methyl transferase (6). Discrimination of
selenium from sulfur is important for establishing the role of selenium
as an essential trace element in mammals and other organisms (7).
SCL of pig liver (8) was characterized as the first enzyme that
specifically acts on a selenium-containing substrate. We have found
that the peptide sequences obtained from the proteolysate of SCL are
similar to those of NifS
proteins.2 However, a gene
encoding SCL has not been cloned yet, and the physiological role of the
enzyme remains to be investigated.
Selenoproteins such as formate dehydrogenase from Escherichia
coli contain selenocysteine residues (9, 10).
Selenocysteyl-tRNASec is required for the biosynthesis of
these selenoproteins (5, 11, 12). Selenophosphate is a highly reactive
selenium compound, and serves as a selenium donor for the
selenocysteyl-tRNASec production (4, 7, 12).
Selenophosphate is synthesized from selenide and ATP, which is
catalyzed by selenophosphate synthetase (13, 14). Recently, Lacourciere
and Stadtman (2) found that the replacement of selenide by NifS and
L-selenocysteine in an in vitro selenophosphate
synthetase assay resulted in an increased rate of formation of
selenophosphate, indicating that selenium derived from
L-selenocysteine by the action of NifS serves as a better
substrate than selenide for selenophosphate synthetase.
In Azotobacter vinelandii, NifS functions in nitrogen
fixation by supplying sulfur to stabilize or repair the Fe-S cluster of
the nitrogenase component protein (15). NifS homologs also occur in
many nondiazotrophic procaryotes, including E. coli (16, 17)
and Bacillus subtilis (18), and in eucaryotes, including Saccharomyces cerevisiae (19), Caenorhabditis
elegans (20), mice (21), and humans.2 These NifS
homologs are proposed to play a general role in the mobilization of
sulfur for Fe-S cluster synthesis (22). However, the exact roles of the
nifS-like genes in these non-nitrogen-fixing organisms have
not been clarified, and it is possible that some of these NifS homologs
act physiologically as selenocysteine-specific enzymes (e.g.
SCL) to facilitate the selenophosphate synthesis, as proposed by
Lacourciere and Stadtman (2).
The E. coli genome contains three genes with sequence
homology to nifS. Two enzymes, IscS (17) and cysteine
sulfinate desulfinase (CSD) (16), among the three NifS homologs have
been isolated and characterized. E. coli IscS, which shows
significant sequence identity (40%) to the A. vinelandii
NifS, can deliver the sulfur from L-cysteine for the
in vitro synthesis of the Fe-S cluster of dihydroxyacid
dehydratase from E. coli (17). CSD exhibits both
selenocysteine lyase and cysteine desulfurase activities in addition to
cysteine sulfinate desulfinase activity, and the enzyme is distinct
from A. vinelandii NifS in its amino acid sequence, absorption spectrum, and lack of cysteine residues catalytically essential for the decomposition of L-selenocysteine (16).
Neither enzyme shows strict specificity for
L-selenocysteine, and both act on L-cysteine.
Thus, we have explored the possibility that the last nifS
homolog (csdB)3
mapped at 37.9 min (23) in the chromosome encodes SCL, which plays a
crucial role in selenophosphate synthesis. We have isolated the gene
product (CsdB), studied its enzymatic properties, and carried out
preliminary x-ray crystallographic studies.
Materials--
Restriction enzymes and other DNA modifying
enzymes were purchased from New England Biolabs (Beverly, MA) and
Takara Shuzo (Kyoto, Japan); molecular weight markers for SDS-PAGE and
gel filtration were from Amersham Pharmacia Biotech (Uppsala, Sweden) and Oriental Yeast (Tokyo, Japan); oligonucleotides were from Biologica
(Nagoya, Japan); Gigapite was from Seikagaku Corporation (Tokyo,
Japan); DEAE-Toyopearl, Phenyl-Toyopearl and Butyl-Toyopearl were from
Tosoh (Tokyo, Japan). L-Selenocystine was synthesized as
described previously (24). L-Selenocysteine was prepared from L-selenocystine according to the previous method (8). The Kohara/Isono miniset clone No. 430 (25) was a kind gift from Dr.
Yuji Kohara, National Institute of Genetics, Japan. All other chemicals
were of analytical grade.
Cloning of the csdB Gene--
The DNA fragment containing
csdB was cloned from the chromosomal DNA of E. coli K-12 ICR130 by polymerase chain reaction in a manner
identical to that used for cloning of csdA (16).
Oligonucleotide primers used were
5'-GGAATTCAGGAGGTGCCATATGATTTTTTCCGTCGAC-3' (upstream) and 5'-CCCAAGCTTATCCCAGCAAACGGTG-3'
(downstream); underlining indicates EcoRI and
HindIII sites, respectively, and bold face indicates a
putative ribosome binding sequence. The polymerase chain reaction
product was ligated into pUC118, and then the resultant expression
plasmid, pCSDB, was introduced into E. coli JM109 competent cells.
Enzyme Assays--
The enzyme was assayed in 0.12 M
Tricine-NaOH buffer at pH 7.5. The enzymatic activities toward
L-selenocysteine and L-cysteine were measured
with lead acetate as described previously (16). The previously reported
value (8) for a molar turbidity coefficient of PbSe at 400 nm was
corrected as 1.18 × 104
M Purification of the csdB Gene Product (CsdB)--
Purification
was carried out at 0-4 °C, and potassium phosphate buffer (KPB) (pH
7.4) was used as the buffer throughout the purification. E. coli JM109 cells harboring pCSDB were grown in 9 liters of LB
medium containing 200 µg/ml ampicillin and 1 mM isopropyl-1-thio- Purification of CSD and IscS--
Purification of CSD from
E. coli JM109 transformed with a plasmid pCSD1 containing
the csdA gene was performed as described previously (16).
Expression and purification of recombinant IscS will be described
elsewhere.4 Briefly, the
iscS gene was amplified by polymerase chain reaction with
the Kohara miniset clone No. 430 (25) as a template and inserted into
the NdeI and HindIII sites in pET21a (Novagen,
Madison, WI) to yield pEF1. IscS was isolated from BL21 (DE3) pLysS
cells harboring pEF1 by sonication, ammonium sulfate fractionation, and
chromatography with Phenyl-Toyopearl, DEAE-Toyopearl, Butyl-Toyopearl, Gigapite, and Superose 12 (Amersham Pharmacia Biotech, Uppsala, Sweden) columns.
Analytical Methods--
Protein was quantified by the Bradford
method (27) using Protein Assay CBB solution (Nacalai Tesque, Kyoto,
Japan) with bovine serum albumin as a standard. The concentration of
the purified enzyme was determined with the value Crystallography--
Crystals of CsdB were grown by the hanging
drop vapor diffusion method. Each droplet was prepared by mixing 5 µl
of 20 mg/ml enzyme in 10 mM KPB (pH 7.4) with an equal
volume of each reservoir solution of the Crystal ScreenTM (Hampton
Research, CA) initially and of a modified reservoir solution
subsequently. The yellow crystals of CsdB were mounted in glass
capillaries with the crystallographic c* axis along the
rotation axis of the spindle and subjected to x-ray experiments. Native
data for structure determination were collected at 20 °C with a
Rigaku R-AXIS IIC imaging plate detector using double focusing
mirror-monochromated CuK Cloning and Expression of the csdB Gene and Purification of the
Product--
For the production of a large amount of CsdB, expression
plasmids were constructed as described under "Experimental
Procedures" with chromosomal DNA isolated from E. coli
K-12. The nucleotide sequence of csdB in the expression
vector (pCSDB) was confirmed to be identical with that registered in
GenBankTM accession number D90811 (open reading frame
o320#17). The clone provided overexpression of the cloned gene: about
10% of the total protein in the extract of E. coli JM109
recombinant cells. In the representative purification (Table
I), about 8 mg of homogeneous preparation
of CsdB was obtained per liter of culture.
Physical Characterization--
CsdB provided a single band
corresponding to the Mr of 43,000 on SDS-PAGE
(Fig. 1). The N-terminal sequence of the
purified enzyme, MIFSVDKVRA, agreed with that deduced from the
nucleotide sequence of csdB. The Mr
of the native enzyme was determined to be 88,000 by gel filtration.
Consequently, the enzyme is a dimer composed of two identical subunits.
The spectrophotometric properties of the enzyme were very similar to
those of CSD with an absorption maximum at 420 nm (Fig.
2) at pH 7.4. No significant changes in the absorption spectrum were observed in the range of pH 6-8. This
absorption peak is characteristic of PLP enzymes, which contain the
cofactor bound to the Catalytic Activity and Substrate Specificity--
CsdB catalyzed
the removal of a substituent at the Crystallization and Preliminary X-ray Characterization--
CsdB
was crystallized at 25 °C within 2 days by hanging drop vapor
diffusion against a 100 mM cacodylate solution (pH 6.8) containing 1.4 M sodium acetate, which corresponds to the
solution No.7 in the Crystal ScreenTM. The crystals were
also obtained in 100 mM KPB (pH 6.8) containing 1.4 M sodium acetate and 10 µM PLP, and these
conditions were further used for the crystallization of the enzyme. The
yellow crystals (0.5 × 0.5 × 0.4 mm3) had
tetragonal-bipyramidal shapes (Fig. 3).
They were grown in amorphous debris, which was removed from the
crystals before they were sealed in thin-walled glass capillaries.
The space group of the CsdB was
P43212 with the cell dimensions of
a = b = 128.1 Å, and c = 137.0 Å. The assumption that a single dimer (89 kDa) exists in the
asymmetric unit of the crystal gives a Vm value
of 3.19 Å3/Da, which is equivalent to a solvent content of
62%. These values lie within the range of values commonly found for
proteins (32). A set of native data was collected to 2.8 Å resolution
on a Rigaku R-AXIS IIC using 1.5° oscillation over a range of 45°
(94.2% complete with 23,770 independent reflections). The
Rmerge value for the intensity data was 7.22%.
The data collection statistics obtained for the native CsdB crystals
are given in Table IV. The x-ray crystal
structure determination of the enzyme is now under way by the multiple
isomorphous replacement method.
We also obtained crystals of CSD at 25 °C by hanging drop vapor
diffusion against a 100 mM sodium acetate solution (pH 4.6) containing 200 mM ammonium acetate and 30% (w/v)
polyethylene glycol 4000. However, these crystals were small and not
suitable for x-ray analysis. Further optimization of crystallization
conditions by changing pH, polyethylene glycol concentration, and salt
has resulted in little improvement.
Comparison with Other PLP-dependent
Enzymes--
Grishin et al. (33) classified PLP enzymes
into seven distinct fold types on the basis of primary structure,
secondary structure prediction, and biochemical function. NifS proteins
have been classified as "aminotransferases class V" in the fold
type I together with serine-pyruvate aminotransferase (EC 2.6.1.51),
phosphoserine aminotransferase (EC 2.6.1.52), isopenicillin N
epimerase, and the small subunit of the soluble hydrogenase. Recently,
three-dimensional structures of phosphoserine aminotransferases from
Bacillus circulans sbsp.
Alkalophilus5 and
E. coli6 were
solved and deposited in the Protein Data Bank, Brookhaven National
Laboratory, with the codes 1BT4 and 1BJN, respectively. Comparison of
the structures of phosphoserine aminotransferases with that of CsdB
will contribute to the understanding of how the related proteins confer
separate reaction specificities on the same coenzyme.
The reaction of CsdB shares some common features with that of other
PLP-dependent enzymes such as aspartate A Possible Role of CsdB in Vivo--
Genome sequencing projects
have revealed that homologs of A. vinelandii nifS are
widespread throughout nature and that some organisms contain more than
one copy of a nifS homolog (16, 22). Some of the
"NifS-like proteins" characterized so far prefer L-cysteine to L-selenocysteine, and some of
them show the opposite preference. Further experiments will need to be
done to determine whether putative NifS-like proteins can play a role
in Fe-S cluster assembly.
Lacourciere and Stadtman (2) have pointed out that in vivo
concentrations of sulfur-containing compounds are on the order of a
thousand times greater than those of their selenium analogs (36). Thus,
enzymes showing higher activity toward L-cysteine, such as
A. vinelandii NifS, will preferentially utilize
L-cysteine over L-selenocysteine in
vivo (2). Therefore, it may be reasonable to assume that enzymes
which are specific toward L-selenocysteine probably
function as a physiological selenide delivery system in E. coli. Although CsdB is not strictly specific to selenocysteine, its discrimination factor (290 times over the activity on cysteine) is
much higher than those of other NifS homologs of E. coli.
Accordingly, the enzyme can be regarded as an E. coli
counterpart of mammalian selenocysteine lyase. It would be particularly
intriguing to determine whether CsdB is more effective than CSD and
IscS as a selenide delivery protein in the formation of selenophosphate
catalyzed by E. coli selenophosphate synthetase. The
in vivo function of CsdB is now being studied by disrupting
its gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1·cm
1, and this value was
used in this study. Sulfite produced from L-cysteine
sulfinic acid was determined with fuchsin (26). Production of alanine
from substrates was determined with a Beckman 7300 high performance
amino acid analyzer (Beckman Coulter, Fullerton, CA). Specific activity
was expressed as units/mg of protein, with 1 unit of enzyme defined as
the amount that catalyzed the formation of 1 µmol of the product in 1 min.
-D-galactopyranoside at 37 °C for
16 h. The cells were harvested by centrifugation, suspended in 10 mM KPB, and disrupted by sonication. The cell debris was
removed by centrifugation, and the supernatant solution was
fractionated by ammonium sulfate precipitation (25-50% saturation).
The enzyme was dissolved in 10 mM KPB and dialyzed against
the same buffer. The enzyme was applied to a DEAE-Toyopearl column
(3 × 15 cm) equilibrated with the same buffer. After the column
was washed with the same buffer, the enzyme was eluted with a 0.8-liter
linear gradient of 0-0.25 M NaCl in the buffer. The active
fractions were pooled (110 ml) and concentrated by ultrafiltration
through an Advantec UP-20 membrane (Advantec, Naha, Japan). The enzyme
was dialyzed against 10 mM buffer containing 0.65 M ammonium sulfate and applied to a Phenyl-Toyopearl column
(3 × 15 cm) equilibrated with the same buffer. The enzyme was
eluted with a 0.7-liter linear gradient of 0.65-0.3 M
ammonium sulfate in the buffer, and the active fractions were pooled
and concentrated as above. The enzyme was dialyzed against 10 mM buffer and applied to a Gigapite column (3 × 10 cm) equilibrated with the same buffer. The enzyme was eluted with a
1-liter linear gradient of 10-150 mM KPB, and the active
fractions were collected and concentrated. The final preparation was
further concentrated with Centriprep-10 (Millipore, Bedford, MA) to a volume of 2.7 ml.
M = 4.8 × 104
M
1·cm
1 at 280 nm, which was
calculated from the content of tyrosine, tryptophan, and cysteine (28).
The subunit and the native Mr of CsdB were
determined by SDS-PAGE (29) and gel filtration with Superdex 200 (Amersham Pharmacia Biotech, Uppsala, Sweden), respectively. The PLP
content of the enzyme was determined fluorometrically with KCN
according to the method of Adams (30).
radiation that was
generated with a 0.3-mm focal cup of an x-ray generator RU300 (Rigaku,
Tokyo, Japan) operated at 40 kV and 100 mA. The crystal-to-detector
distance was set to 130.0 mm. Data reduction was carried out using the
R-AXIS IIC software package.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Purification of CsdB
-amino group of a lysine residue at the active
site. However, CsdB is distinct from either of two A. vinelandii proteins, NifS and IscS, and also from IscS of E. coli, all of which have an absorption maximum around 390 nm (15, 17, 22). Reduction with sodium borohydride resulted in a decrease in
the absorption peak at 420 nm with a concomitant increase in the
absorbance at 335 nm (Fig. 2). This result is consistent with that this
is a PLP enzyme. The PLP content of CsdB was determined to be 1.0 mol
per mol of subunit by the fluorometric method (30).
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Fig. 1.
. SDS-PAGE analysis of the purified
CsdB. Lane 1, Mr standards that
include phosphorylase b, bovine serum albumin, ovalbumin,
carbonic anhydrase, and soybean trypsin inhibitor; lane 2,
purified CsdB (4 µg). Coomassie Brilliant Blue R-250 was used for
staining. Numbers represent Mr of
marker proteins.
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Fig. 2.
Absorption spectra of CsdB. The
conditions were as follows: 10 mM KPB, pH 7.4, 25 °C,
0.64 mg/ml of the enzyme. Curve 1, native enzyme;
curve 2, 1 min after addition of sodium borohydride (1 mM) to the enzyme solution.
-carbon of
L-selenocysteine, L-cysteine, and
L-cysteine sulfinic acid to yield L-alanine.
The production of elemental selenium and elemental sulfur from
L-selenocysteine and L-cysteine, respectively,
in the reaction was confirmed in the same manner as reported previously (31). The optimal pH value for the removal of selenium from L-selenocysteine was between 6.5 and 7.5 in Tricine-NaOH or
Mes buffer. The substrate specificity of the enzyme is summarized in
Table II; L-selenocysteine
was the best substrate followed by L-cysteine sulfinic acid
and L-cysteine, in that order. The specific activity of
CsdB on L-selenocysteine (5.5 units/mg) was comparable with
that of CSD and IscS (Table III) but was
about 7 times lower than that of SCL (37 units/mg) (8). The cysteine desulfurase activity of CsdB was about 2 and 5% of that of CSD and
IscS, respectively, at a substrate concentration of 12 mM (Table III). The specific activity of CsdB for L-cysteine
was about 1/290 of the activity with L-selenocysteine
(Table III). This value is much lower than those of CSD and IscS (Table
III). In contrast with CsdB, A. vinelandii NifS favors
L-cysteine as a substrate over its selenium analog (2).
CsdB acted on L-cystine, L-selenocystine, and
L-aspartic acid, although at extremely low rates (<0.08%
of the rate for L-selenocysteine) (Table II).
Substrate specificity of CsdB
Discrimination of L-selenocysteine from
L-cysteine in the reaction catalyzed by CsdB, CSD, and IscS
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Fig. 3.
Photograph of crystals of CsdB grown by the
hanging drop vapor diffusion method.
Data collection statistics
-decarboxylase (EC 4.1.1.12) (34), kynureninase (EC 3.7.1.3) (35), and SCL. These
enzymes catalyze removal of
-substituent from the substrate to form
alanine. None of their structures have been solved. The solution of the
three-dimensional structure of CsdB would contribute to the
understanding of the mechanisms of these PLP-dependent enzymes.
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ACKNOWLEDGEMENT |
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We thank Dr. Yuji Kohara (National Institute
of Genetics, Mishima, Japan) for providing us with the ordered phage clone No. 430.
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FOOTNOTES |
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* This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to H. M.) and by a Research Grant from the Japan Society for the Promotion of Science (Research for the Future) (to N. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-774-38-3240;
Fax: 81-774-38-3248; E-mail: esaki{at}scl.kyoto-u.ac.jp.
2 H. Mihara, T. Kurihara, T. Yoshimura, and N. Esaki, unpublished results.
3 The symbol, csd, was given to designate the postulated cysteine-selenocysteine-decomposition function of a gene product, although the physiological relevance has not been proved.
4 H. Mihara, T. Kurihara, T. Yoshimura, and N. Esaki, manuscript in preparation.
5 G. Hester, T. N. Luong, M. Moser, and J. N. Jansonius, unpublished results.
6 G. Hester, W. Stark, M. Moser, J. Kallen, Z. Markovic-Housley, and J. N. Jansonius, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SCL, selenocysteine lyase; PLP, pyridoxal 5'-phosphate; KPB, potassium phosphate buffer; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine; NifS, cysteine desulfurase; CSD, cysteine sulfinate desulfinase; Mes, 2-(N-morpholino)ethanesulfonic acid.
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