Biosynthesis of Selenocysteine and Its Incorporation into Protein

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Selenium is an essential micronutrient in the diet of humans and other mammals and it has many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function.. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec), and since little was known about how Sec was biosynthesized, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved in its pathway. We recently established the biosynthetic pathway of Sec in eukaryotes and archeae and are now characterizing the components responsible for its synthesis. During the past year, we have, in collaboration with Markus Wahl and Vadim Gladyshev, crystallized the selenocysteine (Sec) synthase (SecS) which is the enzyme that synthesizes Sec on Sec tRNA. We crystallized SecS at 1.65 angstroms resolution, and interestingly, two SecS monomers interact to generate two active sites around pyridoxal phosphate which is a required cofactor for activity. The two dimers in turn further associate to form a homotetramer. By measuring the activity of SecS towards mechanism-based inhibitors, and from the crystallization studies, we were able to ascertain a detailed reaction mechanism for SecS. In addition, we generated a conditional knockout mouse that targeted the removal of SECp43, a protein involved in the methylation of Um34 on Sec tRNA. This methylase is responsible for the final step in the maturation of Sec tRNA and the resulting mature isoform of Sec tRNA has been shown in our laboratory to be responsible for the expression of the subclass of selenoproteins involved in stress-related phenomena in mammals which are designated as stress-related selenoproteins. Therefore, this conditional knockout mouse should elucidate the role of Secp43 in the methylation process and help us further elucidate the Um34 methylase that we are currently characterizing. The initial conditional Secp43 knockout mouse was capable of expressing a second isoform of Secp43 and thus this protein was not fully abolished and could not be studied in this mouse. We have generated a new conditional knockout Secp43 mouse and are currently characterizing and elucidating the role of the targeted removal of Secp43 on Um34 methylation. Furthermore, we have evidence that a second pathway for the biosynthesis of Sec may be present in mammals. To investigate this possibility, we generated a Sec synthase conditional knockout mouse and are also currently characterizing this mouse. Sec synthase is the enzyme responsible for the synthesis of Sec on phosphoseryl-tRNA[Sec] to yield selenocysteyl-tRNA[Sec] which is presently the only known means of synthesizing Sec in mammalian cells.