Structural and Functional Investigation of a Putative Archaeal Selenocysteine Synthase

Bacterial selenocysteine synthase converts seryl-tRNASec to selenocysteinyl-tRNASec for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNASec or M. jannaschii seryl-tRNASec, and neither substrate was directly converted to selenocysteinyl-tRNASec by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNASec kinase and demonstrated that the enzyme converts seryl-tRNASec to O-phosphoseryl-tRNASec that could constitute an activated intermediate for selenocysteinyl-tRNASec production. MJ0158 also failed to convert O-phosphoseryl-tRNASec to selenocysteinyl-tRNASec. In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNASec with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNASec to selenocysteinyl-tRNASec. These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.