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A Family of S-Methylmethionine-dependent Thiol/Selenol Methyltransferases

ROLE IN SELENIUM TOLERANCE AND EVOLUTIONARY RELATION*
Open AccessPublished:February 26, 1999DOI:https://doi.org/10.1074/jbc.274.9.5407
      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.
      SeCys
      selenocysteine
      PAGE
      polyacrylamide gel electrophoresis
      PCR
      polymerase chain reaction
      kb
      kilobase pair(s)
      bp
      base pair
      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.
      • Shrift A.
      ,
      • Brown T.A.
      • Shrift A.
      ,
      • Läuchli A.
      ,
      • Stadtman T.C.
      ).
      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 (
      • Stadtman T.C.
      ,
      • Hüttenhofer A.
      • Böck A.
      ). 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.
      • Stadtman T.C.
      ). Monoselenophosphate is also the selenium donor for the conversion of 2-thiouridine into 2-selenouridine in several tRNA species (
      • Wittwer A.J.
      • Stadtman T.C.
      ,
      • Stadtman T.C.
      ).
      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.
      • Shrift A.
      ,
      • Brown T.A.
      • Shrift A.
      ,
      • Läuchli A.
      ). 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 (
      • Trelease S.F.
      • DiSomma A.A.
      • Jacobs A.L.
      ,
      • Shrift A.
      • Virupaksha T.K.
      ,
      • Shrift A.
      • Virupaksha T.K.
      ); (ii) an increased tolerance to selenium (
      • Trelease S.F.
      ); and (iii) a greatly reduced incorporation of selenium into cellular proteins (
      • Brown T.A.
      • Shrift A.
      ). 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.
      • Brown T.A.
      • Shrift A.
      ). 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 (
      • Brown T.A.
      • Shrift A.
      ). 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 (
      • Brown T.A.
      • Shrift A.
      ,
      • Virupaksha T.K.
      • Shrift A.
      ,
      • Burnell J.N.
      ). 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 (
      • Neuhierl B.
      • Böck A.
      ).
      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.

      DISCUSSION

      Methylation has long been inferred as a means for selenium detoxification (
      • Ganther H.E.
      ,
      • Spallholz J.E.
      ,
      • Hoffman J.L.
      • McConnell K.P.
      ,
      • Mozier N.M.
      • McConnell K.P.
      • Hoffmann J.L.
      ,
      • Carrithers S.L.
      • Hoffman J.L.
      ), based on the observations that dimethylselenide and trimethylselenonium are the major detoxification products in mammals (
      • Schultz J.
      • Lewis H.B.
      ,
      • McConnell K.P.
      ,
      • Palmer I.S.
      • Gunsalus R.P.
      • Halverson A.W.
      • Olson O.E.
      ) and that the main selenium compound in many selenium-accumulating plants is Se-methylselenocysteine (for a review, see Ref.
      • Brown T.A.
      • Shrift A.
      ). Within this line of evidence, expression of a thiopurine methyltransferase gene from Pseudomonas syringaevery recently was found to confer resistance to tellurite and selenite in E. coli (
      • Cournoyer B.
      • Watanabe S.
      • Vivian A.
      ).
      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.
      • Brown T.A.
      • Shrift A.
      ). 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.
      Figure thumbnail gr7
      Figure 7Proposed 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 fromS-methylmethionine to homocysteine. Such an enzyme activity has been described earlier in extracts from E. coli,Saccharomyces cerevisiae, and jack bean meal (
      • Balish E.
      • Shapiro S.K.
      ,
      • Shapiro S.K.
      • Yphantis D.A.
      • Almenas A.
      ,
      • Shapiro S.K.
      • Almenas A.
      • Thomson J.F.
      ,
      • Abrahamson L.
      • Shapiro S.K.
      ); in a parallel study it was shown to play a role inS-methylmethionine catabolism in E. coli.
      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 (
      • Dev I.K.
      • Harvey R.J.
      )) would be sufficient. The effect of S-adenosylmethionine supplementation could not be tested, since this compound is not taken up by E. coli cells (
      • Holloway C.T.
      • Greene R.C.
      • Su C.-H.
      ).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.
      • Mudd S.H.
      • Datko A.H.
      , 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 (
      • James F.
      • Nolte K.D.
      • Hanson A.D.
      ). It can be detected in the plant vacuole as well as in the chloroplast and the cytoplasm, where sulfur metabolism is localized (
      • Trossat C.
      • Rathinasabapathi B.
      • Weretilnyk E.A.
      • Shen T.-L.
      • Huang Z.-H.
      • Gage D.A.
      • Hanson A.D.
      ). 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 (
      • Wu S.-E.
      • Huskey W.
      • Borchardt R.T.
      • Schowen R.L.
      ,
      • Hoffman J.L.
      ). 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 (
      • Hoffman J.L.
      ).

      Acknowledgments

      We are very grateful to M. H. Zenk and T. Kutchan for helpful discussions.

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