A Family of S-Methylmethionine-dependent Thiol/Selenol Methyltransferases
ROLE IN SELENIUM TOLERANCE AND EVOLUTIONARY RELATION*

Bernhard NeuhierlDagger , Martin ThanbichlerDagger , Friedrich Lottspeich§, and August BöckDagger

From the Dagger  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
Top
Abstract
Introduction
References

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
Top
Abstract
Introduction
References

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, gamma -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.

    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).


View larger version (75K):
[in this window]
[in a new window]
 
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.

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.


View larger version (66K):
[in this window]
[in a new window]
 
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.

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).


View larger version (97K):
[in this window]
[in a new window]
 
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.

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).

                              
View this table:
[in this window]
[in a new window]
 
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

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).

                              
View this table:
[in this window]
[in a new window]
 
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 Delta 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 (Delta 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.

                              
View this table:
[in this window]
[in a new window]
 
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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Selenium detoxification by selenocysteine methyltransferase and by homocysteine methyltransferase. E. coli MTD1 cells (Delta 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 Delta 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.


View larger version (131K):
[in this window]
[in a new window]
 
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 Delta 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.


View larger version (52K):
[in this window]
[in a new window]
 
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.


View larger version (17K):
[in this window]
[in a new window]
 
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.

    REFERENCES
Top
Abstract
Introduction
References
  1. Shrift, A. (1969) Annu. Rev. Plant Physiol. 20, 475-494[CrossRef]
  2. Brown, T. A., and Shrift, A. (1982) Biol. Rev. 57, 59-84
  3. Läuchli, A. (1993) Bot. Acta 106, 455-468
  4. Stadtman, T. C. (1996) Annu. Rev. Biochem. 65, 83-100[CrossRef][Medline] [Order article via Infotrieve]
  5. Hüttenhofer, A., and Böck, A. (1998) in RNA Structure and Function (Simons, R. W., and Grunberg-Manago, M., eds), pp. 603-639, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  6. Wittwer, A. J., and Stadtman, T. C. (1986) Arch. Biochem. Biophys. 248, 540-550[Medline] [Order article via Infotrieve]
  7. Stadtman, T. C. (1990) Annu. Rev. Biochem. 59, 111-127[CrossRef][Medline] [Order article via Infotrieve]
  8. Trelease, S. F., DiSomma, A. A., and Jacobs, A. L. (1960) Science 132, 618[Medline] [Order article via Infotrieve]
  9. Shrift, A., and Virupaksha, T. K. (1963) Biochim. Biophys. Acta 71, 483-485[Medline] [Order article via Infotrieve]
  10. Shrift, A., and Virupaksha, T. K. (1965) Biochim. Biophys. Acta 100, 65-75[Medline] [Order article via Infotrieve]
  11. Trelease, S. F. (1942) Science 95, 656-657
  12. Brown, T. A., and Shrift, A. (1981) Plant Physiol. 67, 1051-1053
  13. Virupaksha, T. K., and Shrift, A. (1965) Biochim. Biophys. Acta 107, 69-80[Medline] [Order article via Infotrieve]
  14. Burnell, J. N. (1981) Plant Physiol. 67, 316-324
  15. Neuhierl, B., and Böck, A. (1996) Eur. J. Biochem. 239, 235-238[Abstract]
  16. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33, 103-109[CrossRef][Medline] [Order article via Infotrieve]
  17. Studier, F. W., and Moffat, B. A. (1986) J. Mol. Biol. 189, 113-130[Medline] [Order article via Infotrieve]
  18. Low, B. (1968) Proc. Natl. Acad. Sci. U. S. A. 60, 160-167[Medline] [Order article via Infotrieve]
  19. Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997) Science 277, 1453-1474[Abstract/Free Full Text]
  20. Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., and Kushner, S. R. (1989) J. Bacteriol. 171, 4617-4622[Medline] [Order article via Infotrieve]
  21. Miller, J. H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Cox, J. C., Edwards, E. S., and DeMoss, J. A. (1981) J. Bacteriol. 145, 1317-1324[Medline] [Order article via Infotrieve]
  24. Kuhn, I., Stephenson, F. H., Boyer, H. W., and Greene, J. (1986) Gene (Amst.) 44, 253-263
  25. Aiba, H., Adhya, S., and de Crombrugghe, B. (1981) J. Biol. Chem. 256, 11905-11910[Abstract/Free Full Text]
  26. Gubler, U. (1988) Nucleic Acids Res. 16, 2726[Medline] [Order article via Infotrieve]
  27. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 262, 1074-1078
  28. Müller, S., Senn, H., Gsell, B., Vetter, W., Baron, C., and Böck, A. (1994) Biochemistry 33, 3404-3412[Medline] [Order article via Infotrieve]
  29. Chang, A. C. Y., and Cohen, S. N. (1987) J. Bacteriol. 134, 1141-1156
  30. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  31. Tanaka, H., and Soda, K. (1987) Methods Enzymol. 143, 240-243[Medline] [Order article via Infotrieve]
  32. Brinkmann, U., Mattes, R. E., and Buckel, P. (1989) Gene (Amst.) 85, 109-114[CrossRef][Medline] [Order article via Infotrieve]
  33. Yasukawa, T., Kanei-Ishii, C., Maekawa, T., Fujimoto, J., Yamamoto, T., and Ishii, S. (1995) J. Biol. Chem. 270, 25328-25331[Abstract/Free Full Text]
  34. Goulding, C. W., Postigo, D., and Matthews, R. G. (1997) Biochemistry 36, 8082-8091[CrossRef][Medline] [Order article via Infotrieve]
  35. Zappia, V., Zydek-Cwick, C. R., and Schlenk, F. (1969) Biochim. Biophys. Acta 178, 185-187[Medline] [Order article via Infotrieve]
  36. Ganther, H. E. (1966) Biochemistry 5, 1089-1098[Medline] [Order article via Infotrieve]
  37. Spallholz, J. E. (1997) Biomed. Environ. Sci. 10, 260-270[Medline] [Order article via Infotrieve]
  38. Hoffman, J. L., and McConnell, K. P. (1987) Arch. Biochem. Biophys. 254, 534-540[CrossRef][Medline] [Order article via Infotrieve]
  39. Mozier, N. M., McConnell, K. P., and Hoffmann, J. L. (1988) J. Biol. Chem. 263, 4527-4531[Abstract/Free Full Text]
  40. Carrithers, S. L., and Hoffman, J. L. (1994) Biochem. Pharmacol. 48, 1017-1024[Medline] [Order article via Infotrieve]
  41. Schultz, J., and Lewis, H. B. (1940) J. Biol. Chem. 133, 199-207
  42. McConnell, K. P. (1942) J. Biol. Chem. 145, 55-60
  43. Palmer, I. S., Gunsalus, R. P., Halverson, A. W., and Olson, O. E. (1970) Biochim. Biophys. Acta 208, 260-266[Medline] [Order article via Infotrieve]
  44. Cournoyer, B., Watanabe, S., and Vivian, A. (1998) Biochim. Biophys. Acta 1397, 161-168[Medline] [Order article via Infotrieve]
  45. Balish, E., and Shapiro, S. K. (1967) Arch. Biochem. Biophys. 119, 62-68[Medline] [Order article via Infotrieve]
  46. Shapiro, S. K., Yphantis, D. A., and Almenas, A. (1964) J. Biol. Chem. 239, 1551-1556[Free Full Text]
  47. Shapiro, S. K., Almenas, A., and Thomson, J. F. (1965) J. Biol. Chem. 240, 2512-2518[Free Full Text]
  48. Abrahamson, L., and Shapiro, S. K. (1965) Arch. Biochem. Biophys. 109, 376-382
  49. Dev, I. K., and Harvey, R. J. (1984) J. Biol. Chem. 259, 8402-8406[Abstract/Free Full Text]
  50. Holloway, C. T., Greene, R. C., and Su, C.-H. (1970) J. Bacteriol. 104, 734-747[Medline] [Order article via Infotrieve]
  51. Mudd, S. H., and Datko, A. H. (1990) Plant Physiol. 93, 623-630
  52. James, F., Nolte, K. D., and Hanson, A. D. (1995) J. Biol. Chem. 270, 22344-22350[Abstract/Free Full Text]
  53. Trossat, C., Rathinasabapathi, B., Weretilnyk, E. A., Shen, T.-L., Huang, Z.-H., Gage, D. A., and Hanson, A. D. (1998) Plant Physiol. 116, 165-171[Abstract/Free Full Text]
  54. Wu, S.-E., Huskey, W., Borchardt, R. T., and Schowen, R. L. (1983) Biochemistry 22, 2828-2832[Medline] [Order article via Infotrieve]
  55. Hoffman, J. L. (1986) Biochemistry 25, 4444-4449[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.