CLOTTING AND FUNCTIONAL-CHARACTERIZATION OF HUMAN SELENOPHOSPHATE SYNTHETASE, AN ESSENTIAL COMPONENT OF SELENOPROTEIN SYNTHESIS

Selenocysteine is co translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons. However, the only c omponent of the eukaryotic selenocysteine incorporation machinery iden tified to date is the selenocysteine-specific tRNA(Sec). In prokaryote s, selenocysteine is synthesized from seryl-tRNA(Sec) and the active s elenium donor, selenophosphate. Selenophosphate is synthesized from se lenide and ATP by the selD gene product, selenophosphate synthetase, a nd is required for selenocysteine synthesis and incorporation into bac terial selenoproteins. We have now cloned human selD and shown that tr ansfection of the human selD cDNA into mammalian cells results in incr eased selenium labeling of a mammalian selenoprotein, type 1 iodothyro nine deiodinase. Despite significant differences between the mechanism s of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring sel enium incorporation into bacterial selenoproteins. Human selenophospha te synthetase has only 32% homology with the bacterial protein, althou gh a highly homologous region that has similarity to a consensus ATP/G TP binding domain has been identified. Point mutations within this reg ion result in decreased incorporation of selenium into type 1 iodothyr onine deiodinase in all but one case. Further analysis revealed that r educed selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases.