A Family of S-Methylmethionine-dependent
Thiol/Selenol Methyltransferases
ROLE IN SELENIUM TOLERANCE AND EVOLUTIONARY RELATION*
Bernhard
Neuhierl
,
Martin
Thanbichler
,
Friedrich
Lottspeich§, and
August
Böck
¶
From the
Lehrstuhl für Mikrobiologie der
Universität München, Maria-Ward-Straße 1a,
D-80638 Munich and § Max-Planck-Institut für
Biochemie, Abteilung Proteinchemie, Am Klopferspitz,
D-82152 Martinsried, Germany
 |
ABSTRACT |
Several plant species can tolerate high
concentrations of selenium in the environment, and they accumulate
organoselenium compounds. One of these compounds is
Se-methylselenocysteine, synthesized by a number of species from the
genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this
organism that catalyzes methyl transfer from
S-adenosylmethionine to selenocysteine. To elucidate the
role of the enzyme in selenium tolerance, the cDNA coding for
selenocysteine methyltransferase from A. bisulcatus was
cloned and sequenced. Data base searches revealed the existence of
several apparent homologs of hitherto unassigned function. The gene for
one of them, yagD from Escherichia coli, was
cloned, and the protein was overproduced and purified. A functional
analysis showed that the YagD protein catalyzes methylation of
homocysteine, selenohomocysteine, and selenocysteine with
S-adenosylmethionine and S-methylmethionine as
methyl group donors. S-Methylmethionine was now shown to be
also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was
set up in E. coli which demonstrated that expression of the
plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided that
S-methylmethionine is present in the medium. It is
postulated that the selenocysteine methyltransferase under selective
pressure developed from an
S-methylmethionine-dependent thiol/selenol methyltransferase.
 |
INTRODUCTION |
Because of the chemical similarity of the elements sulfur and
selenium, many organisms are unable to discriminate between the two in
their metabolism. As a consequence, selenium is processed along the
sulfur pathways and is incorporated unspecifically into low and high
molecular weight compounds normally containing sulfur. The extent of
replacement of sulfur by selenium depends on the ratio of the two
elements in the environment and on the differential affinities of the
sulfur pathway enzymes for their cognate substrate and the
selenium-containing analog (for reviews, see Refs. 1-4).
There are, however, metabolic systems in which biological
discrimination takes place. The first one is the specific synthesis and
insertion of selenocysteine into proteins, directed by a UGA codon in
the respective mRNA (4, 5). Biosynthesis of selenocysteine occurs
in a tRNA-bound state and, therefore, separate from sulfur metabolism.
The crucial step in the discrimination between sulfur and selenium
seems to reside in the synthesis of the selenium donor molecule
monoselenophosphate by the enzyme selenophosphate synthetase (for a
review, see Ref. 4). Monoselenophosphate is also the selenium donor for
the conversion of 2-thiouridine into 2-selenouridine in several tRNA
species (6, 7).
The second biological phenomenon, in which discrimination between
selenium and sulfur occurs is selenium tolerance of plants that
accumulate high amounts of organoselenium compounds (for reviews, see
Refs. 1-3). The majority of these plants belongs to the genus
Astragalus (Fabaceae) and they are characterized by the following: (i) the accumulation of high amounts of selenium, mostly in the form of Se-methylselenocysteine (8-10); (ii) an increased tolerance to selenium (11); and (iii) a greatly reduced incorporation of selenium into cellular proteins (12). Numerous studies
on the specificity of the enzymes in sulfur metabolism of these plants
have shown that they are also involved in the synthesis of
organoselenium compounds (for review, see Ref. 2). A general mechanism
explaining the high selenium tolerance of these plants was not
apparent, however.
A common feature of selenium accumulator plants is that tolerance
is always paralleled by synthesis of selenium-containing compounds like
Se-methylselenocysteine,
-glutamyl-Se-methylselenocysteine, or
selenocystathionine (2). For this reason, it was hypothesized that the
basis of selenium tolerance may reside in the existence of enzymes
scrutinizing the cellular pool of sulfur metabolites for selenium
compounds and converting them to adducts that are non-proteinogenic
(12-14). Indeed, a methyltransferase could be purified recently from a
selenium accumulator species, Astragalus bisulcatus, which specifically methylated selenocysteine
with S-adenosylmethionine as methyl donor. The activity of
this selenocysteine methyltransferase
(SeCys1 methyltransferase)
with L-cysteine was at least 3 orders of magnitude lower
than with L-selenocysteine (15).
In the present communication we present the causal connection between
synthesis of the SeCys-methyltransferase and selenium tolerance. The
cDNA coding for this enzyme in A. bisulcatus has been
cloned and shown to confer selenium tolerance when transferred to
Escherichia coli, provided that the cognate methyl group
donor is available. Moreover, we show that the enzyme belongs to a
class of methyltransferases involved in the metabolism of
S-methylmethionine.
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EXPERIMENTAL PROCEDURES |
Organisms, Media, and Growth Conditions--
E.
coli strain JM109 (16) was used for general cloning purposes and
as a source of chromosomal DNA for amplification of the yagD
gene. SeCys-methyltransferase and YagD were overproduced in E. coli strain BL21(DE3) (17). E. coli strain MTD1,
carrying an in frame deletion in the yagD gene, was
constructed from strain KL19 (18) by deleting nucleotides 270-548 of
the yagD open reading frame (19) according to the method of
Hamilton et al. (20).
A. bisulcatus seeds were from the Western Regional Plant
Introduction Station, Pullman, WA (reference number PI 372510). Seeds were germinated according to Brown and Shrift (12), and seedlings were
grown at 25 °C under continuous illumination. Conditions for plant
cell culture of A. bisulcatus have been described previously (15).
E. coli cells were grown aerobically in LB medium (21) or in
M9 minimal medium (22). Cultures for overproduction of
SeCys-methyltransferase and YagD were grown in overproduction medium
composed of 3% (w/v) tryptone, 1% yeast extract, and 1% NaCl. The
ability of E. coli strains to detoxify selenium in the
presence of S-methylmethionine was tested in M9 minimal
medium containing 0.8% glucose. The concentrations of sulfate and
selenate ions were adjusted to 100 and 50 µM,
respectively. 2-ml cultures with this medium were supplemented with
serial 1:2 dilutions of S-methylmethionine and inoculated in
a ratio of 1:20 (v/v) with cultures that had been adjusted to an
A600 of 0.02. A culture without
S-methylmethionine supplementation was used as a control.
Cells were grown aerobically at 37 °C for 24 h, after which the
A600 was determined. Growth experiments were
performed in at least two independent experiments.
In Vivo Labeling of E. coli Cells with 75Se--
The
in vivo incorporation of 75Se into E. coli under aerobic growth conditions was followed with the method
described by Cox et al. (23), replacing L broth by M9 minimal medium
containing 0.8% glucose and 50 µg/ml chloramphenicol where
appropriate. [75Se]sodium selenite (specific activity, 10 mCi/mmol) was present at 0.3 µM. Cells were harvested by
centrifugation after reaching an A600 of
0.25-0.4 and lysed in SDS sample buffer by heating to 95 °C for 10 min. The supernatant of the subsequent centrifugation (10 min at
14,000 × g and room temperature) was used for
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were dried on
filter paper and autoradiographed overnight on a PhosphorScreen
(Molecular Dynamics, Krefeld, Germany).
Construction of Recombinant Plasmids--
Standard recombinant
DNA techniques were employed according to Sambrook et al.
(22). DNA fragments were amplified by PCR using Goldstar polymerase
(Eurogentech, Belgium) or Pfu polymerase (Stratagene) and
primer concentrations in the range of 0.4-0.6 pmol/µl.
The positive selection vector pUKE was constructed in the following
way: first, a 1.3-kb fragment containing the kanamycin resistance gene
was produced from pUC4 KSAC (Pharmacia, Freiburg, Germany) by
PstI restriction and treatment with Klenow polymerase in the
presence of all four nucleoside triphosphates. It was cloned into
plasmid pUC19 (16), from which the ampicillin resistance gene had been
removed by AvaII/AatII restriction and subsequent treatment with Klenow polymerase in the presence of all four nucleoside triphosphates. The resulting plasmid, containing the promoter of the
kanamycin resistance gene in the opposite orientation with respect to
the lac promoter, was designated pUKB. In the second step,
the sequence coding for the EcoRI restriction endonuclease from plasmid pKGS (24) was amplified by PCR with primers
EcoN (5'-TGTCTAATAAAAAACAGTC-3', identical with nucleotides
2-20 of the EcoRI open reading frame) and EcoC
(5'-TCACTTAGATGTAAGCTG-3', complementary to nucleotides 817-834 of the
open reading frame). The fragment was cloned into pUKB cleaved with
EcoRI and treated with Klenow polymerase. The resulting
plasmid, conferring sensitivity to the presence of 0.5 mM
isopropylthiogalactoside in the medium (24), was named pUKE.
Cloning of the cDNA Coding for Selenocysteine
Methyltransferase from A. bisulcatus--
Total RNA was extracted from
5 g of A. bisulcatus cells using hot acidic phenol as
described by Aiba et al. (25) with the following
modifications: the phenol solution originally described was replaced by
a 1:1 (v/v) mixture of liquefied, unbuffered phenol and an 8 M guanidinium chloride solution containing 0.3 M sodium acetate. After heat treatment (20 min at 65 °C)
and cooling to 4 °C, phase separation was achieved by addition of
0.5 volumes of chloroform, vigorous mixing, and centrifugation. After
repeating the extraction with the aqueous phase, RNA was precipitated
by addition of isopropyl alcohol to a final concentration of 50% (v/v)
and sedimented by centrifugation. Poly(A)+ RNA was enriched
on an oligo(dT)-cellulose matrix (Pharmacia, Freiburg, Germany),
following the protocol provided by the manufacturer. Double-stranded
cDNA was synthesized with the method of Gubler (26). A linker
molecule produced by hybridization of the oligonucleotides Link1
(5'-AAGCTTGGTACCCGGG-3') and 5'-phosphorylated Link2
(5'-AATTCCCGGGTACCAAGCTT-3') was ligated to the cDNA, and molecules
>400 bp were enriched using a "size sep 400" spin column
(Pharmacia, Freiburg, Germany).
The cDNA sequence coding for SeCys-methyltransferase from
A. bisulcatus was cloned by a PCR approach. First,
SeCys-methyltransferase purified from A. bisulcatus (15) was
hydrolyzed with endoprotease Endo-LysC, and peptides were purified and
sequenced by Edman degradation. From these sequences degenerate
oligonucleotides were derived and used as primers for PCR with
cDNA from A. bisulcatus as a template. With primers
38MT34 (5'-ATIGC(AG)TC(AG)TAIGT(CT)TCICC-3'; where I indicates inosine)
and 38MT66/2 (5'-GA(AG)GCICA(AG)GCITA(CT)GC-3'), a 270-bp
fragment could be amplified, cloned into pUC19, and sequenced. From
this sequence, two oligonucleotides with divergent orientation on the
cDNA (38MT-1, 5'-CCATCCTTAGAGGTAAACGC-3' and 38MT-2,
5'-ATCTGATACTTCTGCTTAAG-3') were derived. They were used as primers for
PCR with cDNA that had been hydrolyzed with HindIII and
circularized by ligation at low concentration (<1 µg DNA/ml) as a
template. From this amplification reaction, a 0.5-kb fragment could be
cloned in pUKE and sequenced, which provided approximately 200 bp each
of new sequence information in 5' and 3' direction. Using primers
38MT-2 and 38MT3 (5'-ATTATGTTCTCCTCTTCCAG-3'), PCR amplification was
repeated with non-hydrolyzed, circularized cDNA as a template. A
0.75-kb fragment was cloned into pUKE and sequenced. This provided
information on the 3' end of the cDNA with a putative stop codon,
although a poly(A) sequence was not found. The 5' end of the cDNA
was cloned by first amplifying circularized cDNA with
oligonucleotides 38MT-1 and 38MT-2 as primers. Products >1 kb were
gel-purified and used as templates for a second amplification with
Link2 and 38MT11 (5'-TTGCTTCAAATGCAAGCAGG-3'). From the amplified products, a 0.6-kb DNA fragment could be cloned into pUKE and sequenced, which contained a putative ATG start codon and 57 bp of
5'-untranslated sequence.
Construction of Overexpression Plasmids--
Plasmids for the
overproduction of SeCys-methyltransferase and YagD protein were
constructed from plasmid pT7-7 (27); the open reading frames were
amplified by PCR with Pfu polymerase (primers: 38MT5,
5'-TTACTAGTAGATTTGTTTGC-3' and 38MT8, 5'-ATGTCGTCGCCATTGATAAC-3' for
SeCys-methyltransferase; EcMTN, 5'-ATGTCGCAGAATAATCCG-3' and EcMTC,
5'-TCAGCTTCGCGCTTTTAAC-3' for YAGD) and cDNA from A. bisulcatus or chromosomal DNA from E. coli JM109 for
SeCys-methyltransferase and YagD, respectively. Amplified DNA fragments
were treated with Klenow polymerase in the presence of dATP, dGTP, and
dCTP. They were ligated into pT7-7 which had been cleaved with
NdeI/SmaI and treated with Klenow polymerase
under the same conditions as the PCR fragments. The vector containing
the methyltransferase gene (smtA) from A. bisulcatus was named p7AMT and that carrying the yagD
gene from E. coli was designated p7EMT.
A derivative of pT7-7 containing the trxA gene from E. coli under the control of the T7 promoter was constructed as
follows: the trxA gene was isolated from pSM1 (28) by
EcoRI restriction, treatment with Klenow polymerase, and
restriction with HindIII. pT7-7 was cleaved with
SalI, DNA ends were made flush with Klenow polymerase, the
plasmid was re-cleaved with HindIII, and the trxA fragment was inserted by ligation. The resulting plasmid was named pT77T.
The overexpression plasmid p7AMTT, containing the sequences coding for
SeCys-methyltransferase from A. bisulcatus and
for thioredoxin from E. coli in an artificial operon, was
prepared by transferring the sequence coding for the
SeCys-methyltransferase from p7AMT into pT77T via the XbaI
restriction sites of the plasmids.
Construction of Vectors for Constitutive Expression in E. coli--
Vectors for constitutive production of
SeCys-methyltransferase and YagD in E. coli were constructed
from pACYC184 (29). The plasmid was cleaved with EcoRV and
HincII and blunt-end XbaI fragments from plasmids
p7AMT or p7EMT, containing the open reading frames for
SeCys-methyltransferase or YagD, respectively, were inserted by
ligation so that the corresponding genes could be expressed
constitutively via the tetracycline promoter. The resulting plasmids
were designated pACAMTT and pACEMTT, respectively.
Sequence Comparisons--
Sequences of proteins similar to that
from SeCys-methyltransferase were
obtained via internet from The Institute
of Genomic Research2 and the National
Institutes of Health.3 Sequence alignments were produced
with MegAlign (version 0.97).
SDS-Polyacrylamide Gel Electrophoresis and Immunological
Procedures--
SDS-PAGE was performed according to Laemmli (30).
Crude extracts from plant tissues and cultured cells of A. bisulcatus for SDS-PAGE were obtained by mixing plant material in
a 1:1 ratio (w/v) with SDS sample buffer. The mixture was frozen in
liquid nitrogen, boiled for 10-15 min, and centrifuged. The
supernatant was used for gel electrophoresis. For the generation of a
polyclonal antiserum directed against SeCys-methyltransferase purified
from A. bisulcatus, a rabbit was immunized by intradermal
injection of the protein in a custom immunization program at Eurogentec (Belgium). In immunoblotting experiments (22), the polyclonal anti-SeCys-methyltransferase antiserum was used in a 1:3000-1:5000 dilution employing the enhanced chemiluminescence system from Boehringer Mannheim.
Overproduction and Purification of the YagD Protein--
The
YagD protein was overproduced in E. coli BL21(DE3)
containing the plasmid p7EMT. Cells were grown in 8 liters of
overproduction medium containing 100 µg/ml ampicillin in a 10-liter
laboratory fermenter (Braun, Melsungen, Germany) at 37 °C and
maximal aeration. At an A600 of 1.5, overproduction was started by the addition of isopropylthiogalactoside
to a final concentration of 50 µM. Incubation was
continued until the mass production in the culture had ceased,
thereafter cells were harvested by centrifugation, washed with C-Buffer
(25 mM Tris/Cl, 10 mM magnesium acetate, 1 mM EDTA, and 2 mM dithiothreitol, pH 7.5),
frozen in liquid nitrogen, and stored at
20 °C.
All steps of the purification of YagD were performed at 0-4 °C.
After each step, fractions were analyzed by SDS-PAGE followed by
Coomassie staining, and those containing the YagD protein were pooled.
For the preparation of a crude extract, 20 g of cells were
resuspended in 40 ml of C-buffer containing 1 mM
phenylmethylsulfonyl fluoride and lysed by three passages through a
French pressure cell at 13,000 p.s.i. The soluble (S100) fraction was
obtained by centrifugation (30 min at 30,000 × g,
supernatant = S30, and 2 h at 100,000 × g,
supernatant = S100) and adjusted to 30% ammonium sulfate
saturation. The pellet of the following centrifugation was resuspended
in a small volume of C-buffer and dialyzed overnight against 2 liters
of C-buffer. Proteins were chromatographed on a 1.6 × 10-cm
Q-Sepharose column (Pharmacia, Germany), using a gradient from 0 to 500 mM NaCl in C-buffer over 20 column volumes. YagD was
precipitated overnight from 50% ammonium sulfate, sedimented by
centrifugation, and dissolved in a small volume of C-buffer containing
150 mM KCl. The protein was chromatographed on a 1.6 × 60-cm Superdex 75 gel filtration column (Pharmacia, Germany). Fractions that appeared pure in an SDS gel were pooled, and the YagD
protein was precipitated from 50% ammonium sulfate overnight. For long
term storage, the preparation was dialyzed against 500 ml of C-buffer
containing 50% glycerol and kept at
20 °C.
Overproduction and Purification of Selenocysteine
Methyltransferase--
The purification of SeCys-methyltransferase
from cultured A. bisulcatus cells has been described
previously (15). Overproduction of SeCys-methyltransferase in E. coli was performed with strain BL21(DE3) transformed with plasmids
pUBS520 (31) and p7AMTT as described for the YagD protein. Cells were
lysed, and the soluble fraction was prepared as described for the YagD
protein. The purification protocol for the recombinant protein was
similar to the purification from cultured A. bisulcatus
cells, with the following modifications: after ammonium sulfate
precipitation, hydrophobic interaction chromatography on the
phenyl-Sepharose column was used first, followed by gel filtration on
Superdex 75. Fractions containing SeCys-methyltransferase were
chromatographed on a 1.6 × 10-cm Q-Sepharose column using a
gradient from 0-500 mM KCl in C-buffer over 20 column
volumes. Apparently pure fractions were pooled, whereas fractions
containing contaminating proteins were further purified on a MonoQ
column (Pharmacia, Germany) using the same gradient as before. However,
when elution of SeCys-methyltransferase started, the gradient was held
at the same KCl concentration until all protein was eluted. Fractions
containing pure protein were combined with the pool from Q-Sepharose
chromatography, and SeCys-methyltransferase was precipitated from 70%
ammonium sulfate overnight. For long term storage,
SeCys-methyltransferase was treated as described for the YagD protein.
Enzyme Assays--
The direct assay used for the determination
of SeCys-methyltransferase activity has been described (15). In short,
assays were carried out with purified enzyme in an anaerobic hood under an atmosphere of 97% N2 and 3% H2 to avoid
oxidation of the substrates, which were pre-reduced for at least 30 min
with a 10-fold molar excess of sodium borohydride. Reactions (total
volume of 15 µl) consisted of 50 mM sodium citrate
buffer, pH 6.0, 10 mM magnesium acetate, 2 mM
dithiothreitol, 1 mM EDTA, 1 mM methyl acceptor substrate, and the appropriate amount of protein. After 5 min of
preincubation at 30 °C, the reaction was started by addition of
[methyl-14C]S-adenosylmethionine
(specific activity, 59.3 mCi/mmol, NEN Life Science Products), and
incubation was continued at 30 °C. 2.5-µl samples were withdrawn
from the reaction mixture at the appropriate times, pipetted into 2.5 µl of glacial acetic acid to stop the reaction, and spotted onto
silica gel 60 thin layer chromatography plates in two 2.5-µl
portions. Se-[methyl-14C]selenocysteine was
separated from
[methyl-14C]S-adenosylmethionine by
developing in 1-butanol:acetic acid:H2O = 4:1:1. After
autoradiography, quantification was carried out by densitometry with
the aid of a PhosphorImager (Molecular Dynamics). Calibration was done
by spotting serial dilutions of
[methyl-14C]S-adenosylmethionine
stock solutions onto thin layer chromatography plates and by
autoradiography on the same screen. The same assay was used for the
YagD protein, including 100 µg/ml bovine serum albumin in the
reaction mixture and changing the pH to 6.5.
Reaction velocities with methyl donor substrates other than
[methyl-14C]S-adenosylmethionine
were measured indirectly by [14C]iodoacetamide
derivatization of remaining selenocysteine; reaction mixtures and
conditions were the same as in the assay with
[methyl-14C]S-adenosylmethionine.
At appropriate times, 2.5 µl from the reaction were withdrawn and
transferred into 2.5 µl of a solution containing a 10-fold molar
excess of [14C]iodoacetamide (adjusted to a specific
radioactivity of 0.2 mCi/mmol) and kept at room temperature in the dark
for 30 min. The mixtures were transferred to silica gel thin layer
chromatography plates and treated as described for the
[methyl-14C]S-adenosylmethionine
assay (15). Serial dilutions of selenocysteine were treated likewise
and used as standards. The rates of blank reactions without enzyme were
subtracted to correct for spontaneous oxidation and non-enzymatic
methylation of selenocysteine. As in the direct assay, all experiments
were conducted at least in duplicate.
Chemicals--
L-Selenocysteine was synthesized from
L-3-chloroalanine according to Tanaka and Soda (31) and was
donated by S. Müller (Munich, Germany).
DL-Selenohomocysteine was a gift from A. Holmgren (Stockholm, Sweden). All other chemicals were from commercially available sources.
 |
RESULTS |
Cloning of the cDNA Coding for the Selenocysteine
Methyltransferase from A. bisulcatus--
A newly constructed positive
selection vector (pUKE) was used for cloning of the cDNA sequence
coding for the SeCys-methyltransferase from A. bisulcatus.
It was derived from pUC19 by exchanging the ampicillin resistance gene
for the kanamycin resistance gene from plasmid pUC4KSAC and by ligating
into the multiple cloning site the gene (endo) for the
EcoRI restriction endonuclease, so that its expression was
under the control of the lac promoter. Since production of
the EcoRI enzyme is toxic for E. coli K-12
strains (24), this vector allows direct selection for plasmid molecules that contain an insert in the multiple cloning site 5' of the endo gene.
Using oligodeoxyribonucleotides derived from internal peptide sequences
of the SeCys-methyltransferase and cDNA from A. bisulcatus, a 1.4-kb cDNA containing the gene
(smtA) coding for this protein was cloned by PCR in several
steps (see "Experimental Procedures"). Its nucleotide sequence was
determined (Fig. 1); it codes for a
putative protein of 36.7-kDa molecular mass, which is in good agreement
with the size of 36 and 38 kDa determined for SeCys-methyltransferase under native and denaturing conditions, respectively (15). All the
peptide sequences obtained from the purified protein were present in
the derived amino acid sequence of smtA (Fig. 1).

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Fig. 1.
Sequence of the smtA
cDNA and derived amino acid sequence of the selenocysteine
methyltransferase from A. bisulcatus. Peptide sequences
derived from the enzyme purified from A. bisulcatus are
printed in bold.
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To prove finally that the protein encoded by smtA is
identical to the previously purified SeCys-methyltransferase, the gene was cloned into plasmid pT7-7 and overexpressed. Initially, only a low
overproduction was achieved; it could be significantly augmented by
introducing the plasmid pUBS520, which codes for tRNAArg
from E. coli (32). Its coexpression apparently facilitates the decoding of the six AGA codons of the smtA mRNA. The
amount of SeCys-methyltransferase produced in a soluble form could be further increased by co-production of E. coli thioredoxin
(33) from the same plasmid (p7AMTT). The overproduction level achieved was 10-15% of the total protein, and about 60% of the protein was
soluble (data not shown). The recombinant protein was purified in a way
similar to the protocol employed for the enzyme isolated from plant
cells (15). About 30 mg of purified protein were obtained from 25 g of cells (Fig. 2A). The
recombinant enzyme catalyzed the identical reaction but with a 1.6-fold
higher specific activity, which is probably due to the higher initial
enzyme concentration in the E. coli cells, since the enzyme
loses activity upon dilution (Ref. 15 and data not shown). One can
conclude, therefore, that smtA from A. bisulcatus
codes for SeCys-methyltransferase and that the activity of the enzyme
does not depend on any eucaryote-specific modification, like
glycosylation.

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Fig. 2.
Overproduction and purification of the
Astragalus selenocysteine methyltransferase and of the
YagD protein. Proteins from purification steps were separated by
12.5 (A) or 10% (B) SDS-PAGE and visualized by
Coomassie staining. A, purification of
SeCys-methyltransferase after overproduction; lane 1, S30;
lane 2, S100; lane 3, ammonium sulfate
precipitate (35-60% saturation); lane 4, hydrophobic
interaction chromatography; lane 5, gel filtration;
lane 6, anion exchange chromatography (Q-Sepharose);
lane 7, anion exchange chromatography (MonoQ). B,
overproduction and purification of the YagD protein; lane
1, SDS lysate of cells before induction; lane 2, SDS lysate of cells 3 h after induction; lane 3, S30;
lane 4, S100; lane 5, ammonium sulfate
precipitate (0-30% saturation); lane 6, anion exchange
chromatography; lane 7, gel filtration.
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A data base search for sequences displaying similarity to that of
SeCys-methyltransferase from A. bisulcatus gave a number of
entries for proteins with unassigned function. They are aligned in Fig.
3. The degree of identity ranges from 28 to 30% (YPL273W and YLL062C from yeast) over approximately 40% (YbgG
from Bacillus subtilis, YagD from E. coli, and
Rv2458 from Mycobacterium tuberculosis) to 52-71% for two
"expressed sequence tags" from Oryza sativa and
Arabidopsis thaliana, respectively. There are three strongly conserved motifs in the C-terminal part of the molecules, namely G(I/V)NC, YPNSGE, and GGCCR (Fig. 3). A motif very similar to the
latter one has already been shown to be present in all
cobalamin-dependent methionine synthases and in
betaine:homocysteine methyltransferases. It has been speculated that
residues of this motif might be involved in the binding and/or
activation of homocysteine (34).

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Fig. 3.
Alignment of the sequence of
selenocysteine methyltransferase with those of similar proteins.
SeCys-MT, selenocysteine methyltransferase; Abi,
A. bisulcatus; Bsu, B. subtilis;
Eco, E. coli; Mtu, M. tuberculosis; Sce, S. cerevisiae;
Ath, A. thaliana; Osa, O. sativa; EST, expressed sequence tag. Residues conserved
in all eight sequences are boxed.
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The data base search also revealed a marginal relationship (15-20%
identity) to the N-terminal segment of cobalamin-dependent methionine synthases, e.g. the metH gene product
from E. coli. It is relevant that a segment of MetH
comprising amino acids 2-353, which is approximately the size of the
SeCys-methyltransferase, is still able to catalyze methyl transfer from
methylcobalamin to homocysteine (34), which means that it must contain
the homocysteine binding and methylation site.
Overproduction of the yagD Gene Product from E. coli--
The
considerable sequence identity between the SeCys-methyltransferase from
A. bisulcatus and enzymes from selenium non-accumulating organisms, like the E. coli yagD gene product, raised the
question on the function and physiological role of these unassigned
gene products. The hitherto putative YagD protein from E. coli was chosen for this analysis.
To this end, the yagD coding sequence (19) was amplified by
PCR using oligodeoxyribonucleotides EcMTN and EcMTC as primers and
chromosomal DNA from E. coli strain JM109. The PCR product was cloned into plasmid pT7-7. Overexpression in E. coli
BL21(DE3) led to a soluble gene product comprising about 20% of the
total cellular proteins (Fig. 2B). The recombinant protein
was purified employing the steps detailed in Fig. 2B. The
yield was 50 mg of apparently homogenous protein per 20 g of
cells. Its molecular mass was 35 kDa as judged by SDS-PAGE, which is in
agreement with an expected size of 33.5 kDa derived from the gene
sequence. Elution from the gel filtration column was in the position of
a 34-kDa protein, indicating that YagD in solution is a monomer.
Table I gives the results of an analysis
of the substrate spectrum of the purified YagD protein from E. coli. It shows that the YagD protein catalyzes methyl transfer
from S-adenosylmethionine to homocysteine,
selenohomocysteine, and, although less efficiently, selenocysteine. The
selenium form of the acceptor substrate is methylated 2-3-fold faster
than the sulfur analog. Reaction velocities with L-cysteine
were close to the detection limit. Significant product inhibition by
methionine and S-adenosyl-homocysteine was only observed at
concentrations higher than 0.5 and 0.25 mM, respectively (data not shown).
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Table I
Relative methyltransferase activity of the YagD protein from E. coli in
the direct assay with [methyl-14C]-S-adenosylmethionine as
methyl donor and with different methyl acceptor substrates
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|
The spectrum of methyl donor substrates was determined for both the
recombinant SeCys-methyltransferase and the YagD protein from E. coli. Of the substances tested, namely
S-adenosylmethionine, S-methylmethionine,
trimethylsulfonium, and glycine betaine, both enzymes only accepted
S-adenosylmethionine and S-methylmethionine in
the semi-quantitative assay described previously (15). Since no
methyl-14C-derivative of
S-methylmethionine is available, reaction velocities of
SeCys-methyltransferase were measured by
[14C]iodoacetamide derivatization of the selenocysteine
remaining in the reaction mixture. Due to the low reactivity of
homocysteine in the derivatization reaction, this assay could not be
used for the YagD protein, so that kinetic data for the different
methyl donor substrates are available for SeCys-methyltransferase only (Table II).
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Table II
Activities of selenocysteine methyltransferase from A. bisulcatus with
different methyl donor substrates
Activity with [methyl-14C]AdoMet was measured with
the direct assay, all other methyl donor substrates were assayed
indirectly. SeCys concentration was 1 mM in all reactions.
|
|
Surprisingly, the maximal conversion with
[methyl-14C]S-adenosylmethionine
was only 10% of the substrate input (Table II), a value that also was
found for the YagD protein, compared with 20-25% for unlabeled
S-adenosylmethionine. 100% conversion into product,
however, was obtained with L-S-methylmethionine
as donor. Since the group transfer potentials of
S-methylmethionine and S-adenosylmethionine are
very similar, one may conclude that the enzyme only uses an impurity
present in the S-adenosylmethionine preparation. It is
noteworthy that the non-radioactive
S-adenosyl-L-methionine employed contains about
18% of the non-physiological S(+)-form (with respect to the
sulfur atom; Sigma, product information) which agrees reasonably well
with the maximal conversion (Table II). Furthermore, an enriched Hcy
methyltransferase from yeast was shown to be more active with the
racemic mixture of the two S-adenosylmethionine
stereoisomers than with the R(
) isomer alone (35).
However, since our data base search showed that yeast apparently
possesses two Hcy methyltransferases, it is not possible to attribute
the substrate specificities clearly, so that positive information on
the nature of the actual methyl donor in the
S-adenosyl-L-methionine preparations used is
lacking. Nevertheless, it can be concluded that the YagD protein is an
enzyme that catalyzes methyl transfer from
S-methylmethionine to homocysteine, so it will be designated homocysteine (Hcy) methyltransferase below.
The Role of Selenocysteine Methyltransferase and Homocysteine
Methyltransferase in Selenium Detoxification--
Table
III shows a comparison of the Hcy
methyltransferase from E. coli with the
SeCys-methyltransferase from A. bisulcatus (15). It is
apparent that the two enzymes are related concerning the reaction
catalyzed but differ in their discrimination between sulfur- and
selenium-containing substrates. By this, they provide a unique
experimental model for the detoxification of selenium-containing compounds in a heterologous genetic background. For this purpose, an
E. coli
yagD strain (MTD1) was constructed and
transformed with plasmid pACAMTT or pACEMTT on which the
smtA gene or yagD gene, respectively, is
expressed from the constitutive tet promoter of plasmid
pACYC184. The transformants were grown in minimal medium with a ratio
of sulfate:selenate concentration of 2:1 and varying S-methylmethionine concentrations (Fig.
4), conditions under which growth of
E. coli MTD1 (
yagD) transformed with pACYC184
is inhibited (data not shown). It is evident that synthesis of the
SeCys-methyltransferase from A. bisulcatus provides a much
better protection against the toxic effect of selenate than that of the
Hcy methyltransferase from E. coli. There is also a direct
correlation between the amount of methyl donor compound in the medium
and the obtainable cell yield.
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Table III
Kinetic constants for selenocysteine methyltransferase from A. bisulcatus and the YagD protein from E. coli with different methyl
acceptor substrates
Reaction velocities were measured with the direct assay using
[methyl-14C]S-adenosylmethionine as
methyl
donor.
|
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Fig. 4.
Selenium detoxification by selenocysteine
methyltransferase and by homocysteine methyltransferase. E. coli MTD1 cells ( yagD) constitutively producing
SeCys-methyltransferase (SeCys-MT, from plasmid pACAMTT) or
Hcy methyltransferase (Hcy-MT, from plasmid pACEMTT) were
grown in minimal medium containing 100 µM sulfate, 50 µM sodium selenate, and increasing concentrations of
S-methylmethionine. After 24 h, the cell mass in the
cultures was determined by measuring the optical density at a
wavelength of 600 nm.
|
|
To characterize further the methyltransferase-producing strains, a
[75Se]selenium incorporation experiment was carried out
employing E. coli KL19 wild type, the
yagD
mutant MTD1, and MTD1 transformed with the vector pACYC184 or the
plasmids pACAMTT (smtA) or pACEMTT (yagD). Cell
lysates were separated by SDS-PAGE and autoradiographed (Fig.
5). Presence of plasmids pACAMTT and
pACEMTT prevented cellular proteins from being labeled with
[75Se]selenium but only when
S-methylmethionine was provided in the medium.

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Fig. 5.
Incorporation of 75Se by E. coli cells. Cells were grown aerobically in the
presence of 0.3 µM
[75Se]Na2SeO3 and 0.5 mM S-methylmethionine. SDS lysates of cells were
analyzed by 10% SDS-PAGE. Lane 1, E. coli KL19 (wild type); lane 2, MTD1 (KL19
yagD); lane 3, MTD1/pACYC184; lane
4, MTD1/pACAMTT (smtA); no
S-methylmethionine added; lane 5, MTD1/pACAMTT
(smtA); lane 6, MTD1/pACEMTT
(yagD).
|
|
Expression of smtA in Cell Cultures and in Plant Tissues of A. bisulcatus--
Finally, it was of interest to analyze the time course
of synthesis of SeCys-methyltransferase in cell cultures and the tissue specificity of expression in whole plants. Immunoblotting of SDS lysates separated by SDS-PAGE was employed to quantitate the amount of
material cross-reacting with anti-SeCys-methyltransferase antiserum. Cell cultures contained the maximal level of SeCys-methyltransferase under conditions of exponential growth; stationary phase cells possessed a much lower amount. The presence of selenite in the medium,
however, did not influence synthesis and/or stability (data not shown).
To analyze tissue localization of SeCys-methyltransferase,
4-8-week-old seedlings of A. bisulcatus were separated in
roots, cotyledons, hypocotyl, and primary leaves. SDS lysates were
prepared and the proteins separated by SDS-PAGE and subjected to
immunoblotting analysis (Fig. 6).
SeCys-methyltransferase was present in all plant tissues. Intriguingly,
cotyledons and leaves contained a second cross-reacting band with
faster migration, indicating a size decrease of 1-2 kDa. Since this
shortened form is present only in the green tissues of the plant, it
seems probable that it represents a form that is localized in
chloroplasts.

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Fig. 6.
Localization of selenocysteine
methyltransferase in tissues of A. bisulcatus. Proteins from
tissues of A. bisulcatus were separated by 10% SDS-PAGE and
analyzed for their content of SeCys-methyltransferase by immunoblotting
with anti-SeCys-methyltransferase antiserum (1:3000 dilution).
Lane 1, SeCys-methyltransferase, purified after
overproduction in E. coli (25 ng); lane 2, crude
extract from A. bisulcatus cells; lane 3, A. bisulcatus, roots; lane 4, A. bisulcatus, hypocotyl; lane 5, A. bisulcatus, cotyledons; lane 6, A. bisulcatus, leaves. Lanes 2-6 contained 20-30 µg of
protein each.
|
|
 |
DISCUSSION |
Methylation has long been inferred as a means for selenium
detoxification (36-40), based on the observations that
dimethylselenide and trimethylselenonium are the major detoxification
products in mammals (41-43) and that the main selenium compound in
many selenium-accumulating plants is Se-methylselenocysteine (for a review, see Ref. 2). Within this line of evidence, expression of a
thiopurine methyltransferase gene from Pseudomonas syringae very recently was found to confer resistance to tellurite and selenite
in E. coli (44).
A scheme of our present view on the mechanism of selenium
detoxification by SeCys-methyltransferase is presented in Fig.
7. It is established that selenium is
metabolized along the sulfur pathway, resulting in the synthesis of
selenocysteine as the primary organoselenium compound (for a review,
see Ref. 2). Selenocysteine is methylated with high efficiency by
SeCys-methyltransferase, thus preventing the flux of selenium into
proteins and other sulfur-containing compounds. It is plausible to
assume that Se-methylselenocysteine is transported into the plant
vacuole as a dead-end product; however, conclusive experiments on this
are still lacking.

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Fig. 7.
Proposed roles of selenocysteine
methyltransferase and of homocysteine methyltransferase (YagD) in
sulfur/selenium metabolism. THF, tetrahydrofolic
acid.
|
|
An intriguing result of the sequence analysis of
SeCys-methyltransferase was that the data base search revealed a number
of related sequences with unassigned function. One of the similar proteins, YagD from E. coli, was purified from an
overproducing strain and shown to catalyze methyl transfer from
S-methylmethionine to homocysteine. Such an enzyme activity
has been described earlier in extracts from E. coli,
Saccharomyces cerevisiae, and jack bean meal (45-48); in a
parallel study it was shown to play a role in S-methylmethionine catabolism in E. coli.4 YagD exhibited
only a slight preference for selenohomocysteine compared with
homocysteine as substrate; selenocysteine was methylated with low
efficiency, and methylation of cysteine was below detection limit.
The enzyme from the plant displays 40% sequence identity to YagD from
E. coli; it is, however, almost fully specific for the selenium analogs of cysteine and homocysteine. Certainly, the two
proteins are evolutionarily related, and it is most probable that the
detoxifying SeCys-methyltransferase has evolved from an enzyme not
discriminating between sulfur and selenium substrate analogs. A certain
level of selenium tolerance was already apparent when the YagD protein
was overproduced; it will be interesting to see whether the change of
specificity can be achieved by a mutational approach.
The specificity for S-methylmethionine (and possibly the
S(+) isomer of S-adenosylmethionine) is unusual
for a methyltransferase, but the biochemical evidence is corroborated
by the fact that in vivo selenium detoxification by both
SeCys-methyltransferase and Hcy methyltransferase was directly
dependent on supplementation of S-methylmethionine to the
medium (Fig. 4). This also indicates that
S-adenosylmethionine at intracellular concentrations cannot serve as an effective substrate for both enzymes, although its concentration in E. coli cells (26 µM (49))
would be sufficient. The effect of S-adenosylmethionine
supplementation could not be tested, since this compound is not taken
up by E. coli cells (50).4
This substrate specificity of the YagD protein is plausible, since
otherwise cells producing this enzyme would enter a shortened version
of the futile cycle described by Ref. 51, i.e. synthesis of
S-adenosylmethionine from methionine and ATP, transfer of
the activated methyl group to homocysteine to produce methionine and S-adenosyl-homocysteine, which would subsequently be
hydrolyzed to adenosine and homocysteine. The balance of this cycle
would be hydrolysis of ATP to adenosine, pyrophosphate, and
Pi, without apparent benefit for the cell.
Thus it appears that S-methylmethionine is the main
substrate both for SeCys-methyltransferase from A. bisulcatus and for YagD from E. coli. This compound is
present in many plants in concentrations ranging from 0.01 to 6 µmol
per g dry weight (52). It can be detected in the plant vacuole as well
as in the chloroplast and the cytoplasm, where sulfur metabolism is
localized (53). So, whereas the function of the SeCys-methyltransferase
lies in the detoxification of selenium in selenium-accumulating plants, its homologs seem to play a role in the catabolism of
S-methylmethionine in plants and bacteria (Fig. 7).
Furthermore, an additional role for this class of enzymes in the
consumption of the unphysiological S(+) stereoisomer of
S-adenosylmethionine seems possible; this isomer has been
shown to arise from spontaneous racemization at the sulfur atom (54,
55). Its concentration in mouse liver extracts, however, was
significantly lower than expected from racemization rates, which led to
the speculation that an enzyme activity degrading or utilizing this
substance should exist (55).
 |
ACKNOWLEDGEMENTS |
We are very grateful to M. H. Zenk and
T. Kutchan for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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.
2
Information available on-line at the following
address: http://www.tigr.org.
3
Information available on-line at the following
address: http://www.ncbi.nlm.nih.gov.
4
M. Thanbichler, B. Neuhierl, and A. Böck,
unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
SeCys, selenocysteine;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
kb, kilobase pair(s);
bp, base pair.
 |
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