Structural and functional investigation of a putative archaeal selenocysteine synthase.
Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) 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-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). 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.
Kaiser
Jens T
JT
Division of Chemistry and Chemical Engineering, California Institute of Technology, m/c 114-96, Pasadena, California 91125, USA.
Gromadski
Kirill
K
Rother
Michael
M
Engelhardt
Harald
H
Rodnina
Marina V
MV
Wahl
Markus C
MC
D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D001665:Binding Sites; D003001:Cloning, Molecular; D003101:Collodion; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D020745:Genome, Archaeal; D017017:Methanococcus / Q000201:enzymology; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D055672:Static Electricity; D013499:Surface Properties; D014166:Transferases / Q000737:chemistry*
D000838:Anions; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; C112705:selenocysteinyl-tRNA; D012694:Serine; D003101:Collodion; D014166:Transferases; C060600:selenium transferase