Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation.
Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNA(Sec) synthesis in which O-phosphoseryl-tRNA(Sec) kinase (PSTK) phosphorylates Ser-tRNA(Sec) to produce the O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNA(Sec). This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNA(Sec) formation (K(m) for Ser-tRNA(Sec) of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNA(Sec) and tRNA(Sec) with high affinity (K(d) values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNA(Sec) specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its 'DxTN' kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species.
Sherrer
R Lynn
RL
Department of Molecular Biophysics, Yale University, New Haven, CT 06520-8114, USA.
O'Donoghue
Patrick
P
Söll
Dieter
D
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019143:Evolution, Molecular; D007700:Kinetics; D017015:Methanococcales / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D016415:Sequence Alignment; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity
D019843:Archaeal Proteins; D012346:RNA, Transfer, Amino Acyl; C112705:selenocysteinyl-tRNA; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000251:Adenosine Triphosphatases