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* 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.
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 fromS-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 withS-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 thatS-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from anS-methylmethionine-dependent thiol/selenol methyltransferase.
polyacrylamide gel electrophoresis
polymerase chain reaction
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.
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 (
). 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.
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.
). The majority of these plants belongs to the genusAstragalus (Fabaceae) and they are characterized by the following: (i) the accumulation of high amounts of selenium, mostly in the form of Se-methylselenocysteine (
). 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, γ-glutamyl-Se-methylselenocysteine, or selenocystathionine (
). 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 (
). 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 (
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 toEscherichia 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 ofS-methylmethionine.
Methylation has long been inferred as a means for selenium detoxification (
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.
). 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.
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 fromS-methylmethionine to homocysteine. Such an enzyme activity has been described earlier in extracts from E. coli,Saccharomyces cerevisiae, and jack bean meal (
M. Thanbichler, B. Neuhierl, and A. Böck, unpublished results.
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 fromE. 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 theS(+) 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 thatS-adenosylmethionine at intracellular concentrations cannot serve as an effective substrate for both enzymes, although its concentration in E. coli cells (26 μm (
, i.e. synthesis ofS-adenosylmethionine from methionine and ATP, transfer of the activated methyl group to homocysteine to produce methionine andS-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 (
). 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 ofS-methylmethionine in plants and bacteria (Fig. 7). Furthermore, an additional role for this class of enzymes in the consumption of the unphysiological S(+) stereoisomer ofS-adenosylmethionine seems possible; this isomer has been shown to arise from spontaneous racemization at the sulfur atom (
). 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 (