Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing seleno
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© Springer-Verlag 1998
O R I G I N A L PA P E R
Reinhard Wilting · Kalliopi Vamvakidou · August Böck
Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase
Received: 8 July 1997 / Accepted: 3 September 1997
Abstract The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this selfdependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selDHI) in an E. coli ∆selD strain. Gene selDHI gave rise to a selenium-containing gene product and also supported – via its activity – the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGASec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif. Key words Selenophosphate · Selenoproteins
Introduction Monoselenophosphate is the selenium donor compound for the biosynthesis of selenocysteine (Veres et al. 1992; Glass et al. 1993). Selenomonophosphate formation is catalysed by the enzyme monoselenophosphate synthetase via the selenide-dependent cleavage of ATP, in which the γ-phosphate moiety of ATP is transferred to the selenide anion, and AMP and the β-phosphate are released (Leinfelder et al. 1990; Ehrenreich et al. 1992; Veres et al. 1994; Mullins et al. 1997). A detailed enzymological (Leinfelder et al. 1990) and mutational analysis of the en-
zyme from Escherichia coli (the selD gene product) highlighted the involvement of a cysteine residue (Cys-17) in the reaction mechanism (Kim et al. 1993). The characterisation of the genes coding for selenophosphate synthetases and of their products in Haemophilus influenzae, Methanococcus jannaschii, mouse, and humans has revealed that the enzymes from these organisms are selenoproteins themselves since they carry a selenocysteine residue in the position of the E. coli Cys-17 (Fleischmann et al. 1995; Bult et al. 1996; Guimeraes et al. 1996; Wilting et al. 1997). Thus, the product of the reaction, selenophosphate, is required for the synthesis of the enzyme. This intriguing and unique situation poses the questions (1) how the selenium status of a cell influences the efficiency of selenophosphate synthesis and, as a consequence, selenoprotein formation, and (2) how a cell recovers from selenium starvation in the absence of selenophosphate synthetase activity. This is especially relevant also for eukaryal organisms since it has been shown that inadequate selenium supply causes almost
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