Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

Abstract

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA^[Ser]Sec as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA^[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA^[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. Biosynthesis of the 20 canonical amino acids is well established in eukaryotes. However, many eukaryotes also have a rare selenium-containing amino acid, selenocysteine, which is the 21st amino acid in the genetic code. Selenium is essential for human health, and its health benefits, including preventing cancer and heart disease and delaying aging, have been attributed to the presence of selenocysteine in protein. How selenocysteine is made in eukaryotes has not been established. To gain insight into its biosynthesis, we used computational analyses to search completely sequenced genomes for proteins that occur exclusively in organisms that utilize selenocysteine. This approach revealed a putative selenocysteine synthase, which had been previously identified as a pyridoxal phosphate–containing protein dubbed soluble liver antigen. We were able to characterize the activity of this synthase using selenophosphate and a tRNA aminoacylated with phosphoserine as substrates to generate selenocysteine. Moreover, identification of selenocysteine synthase allowed us to delineate the entire pathway of selenocysteine biosynthesis in mammals. Interestingly, selenocysteine synthase is present only in those archaea and eukaryotes that make selenoproteins, indicating that the newly defined pathway of selenocysteine biosynthesis is active in these domains of life.