Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

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Abstract
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 ( Km 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 ( Kd 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.