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

Sherrer RL, O'Donoghue P, Söll D - Nucleic Acids Res. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Yale University, New Haven, CT 06520-8114, USA.

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 (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.

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In vitro conversion of Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK. (A) PSTK assay with 32P-labeled tRNA. Representative phosphorimage of the separation of Ser-[32P]AMP, [32P]AMP and Sep-[32P]AMP by PEI cellulose chromatography is shown. 1 µM 32P-labeled tRNASec was incubated with 600 nM SerRS (lane 1) or 600 nM SerRS and 100 nM PSTK (lane 2) at 37°C for 45 min. Aliquots of the reactions were quenched with 100 mM sodium citrate, pH 5.0 and digested with 0.66 mg/ml nuclease P1 for 35 min at room temperature. Samples were then spotted onto a PEI-cellulose TLC plate and developed in 100 mM ammonium acetate, 5% acetic acid for 75 min. (B) PSTK assay using [14C]Ser. Phosphorimage of the separation of [14C]Ser-AMP and [14C]Sep-AMP on a PEI-cellulose TLC plate is shown. 2 µM tRNASec was incubated with 1.2 µM SerRS (lane 1) or 1.2 µM SerRS and 200 nM PSTK (lane 2) in the presence of 100 µM [14C]Ser for 45 min at 37°C. The aa-tRNASec products were purified as described in the Materials and Methods section, digested with nuclease P1 and chromatographed as above. (C) PSTK assay with γ-[32P]ATP as the phosphate donor. Phosphorimage of [32P]Sep-AMP on PEI-cellulose is shown. 1 µM Ser-tRNASer (lane 1) or 1 µM Ser-tRNASec (lane 2) was incubated with 100 nM PSTK with 1.67 µM γ-[32P]ATP for 45 min at 37°C. The reactions were purified over a Sephadex G25 Microspin column to remove unincorporated γ-[32P]ATP prior to digestion with nuclease P1 and TLC analysis as in (A).
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Figure 1: In vitro conversion of Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK. (A) PSTK assay with 32P-labeled tRNA. Representative phosphorimage of the separation of Ser-[32P]AMP, [32P]AMP and Sep-[32P]AMP by PEI cellulose chromatography is shown. 1 µM 32P-labeled tRNASec was incubated with 600 nM SerRS (lane 1) or 600 nM SerRS and 100 nM PSTK (lane 2) at 37°C for 45 min. Aliquots of the reactions were quenched with 100 mM sodium citrate, pH 5.0 and digested with 0.66 mg/ml nuclease P1 for 35 min at room temperature. Samples were then spotted onto a PEI-cellulose TLC plate and developed in 100 mM ammonium acetate, 5% acetic acid for 75 min. (B) PSTK assay using [14C]Ser. Phosphorimage of the separation of [14C]Ser-AMP and [14C]Sep-AMP on a PEI-cellulose TLC plate is shown. 2 µM tRNASec was incubated with 1.2 µM SerRS (lane 1) or 1.2 µM SerRS and 200 nM PSTK (lane 2) in the presence of 100 µM [14C]Ser for 45 min at 37°C. The aa-tRNASec products were purified as described in the Materials and Methods section, digested with nuclease P1 and chromatographed as above. (C) PSTK assay with γ-[32P]ATP as the phosphate donor. Phosphorimage of [32P]Sep-AMP on PEI-cellulose is shown. 1 µM Ser-tRNASer (lane 1) or 1 µM Ser-tRNASec (lane 2) was incubated with 100 nM PSTK with 1.67 µM γ-[32P]ATP for 45 min at 37°C. The reactions were purified over a Sephadex G25 Microspin column to remove unincorporated γ-[32P]ATP prior to digestion with nuclease P1 and TLC analysis as in (A).

Mentions: First, we 32P-labeled the terminal 3′-AMP of the tRNA using the exchange reaction of the E. coli CCA-adding enzyme in the presence of [α-32P]ATP. The radioactive tRNA was then serylated by pure M. maripaludis SerRS. After conversion of the Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK, the aa-tRNA products were digested by nuclease P1. The resulting mixture of Sep-[32P]AMP, [32P]AMP and Ser-[32P]AMP was separated on polyethyleneimine (PEI)-cellulose plates by TLC (Figure 1A), giving Rf values of 0.29, 0.50 and 0.85, respectively. This assay allows direct monitoring of the deacylation of the substrate Ser-tRNASec and the product Sep-tRNASec that inevitably occurs over the course of the reaction. We should note that in this study we used the transcript of the M. maripaludis tRNASec; while SerRS serylated both M. jannaschii Ser-tRNASec and M. maripaludis Ser-tRNASec to 70–80%, M. jannaschii Ser-tRNASec deacylated more rapidly than M. maripaludis Ser-tRNASec.Figure 1.


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

Sherrer RL, O'Donoghue P, Söll D - Nucleic Acids Res. (2008)

In vitro conversion of Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK. (A) PSTK assay with 32P-labeled tRNA. Representative phosphorimage of the separation of Ser-[32P]AMP, [32P]AMP and Sep-[32P]AMP by PEI cellulose chromatography is shown. 1 µM 32P-labeled tRNASec was incubated with 600 nM SerRS (lane 1) or 600 nM SerRS and 100 nM PSTK (lane 2) at 37°C for 45 min. Aliquots of the reactions were quenched with 100 mM sodium citrate, pH 5.0 and digested with 0.66 mg/ml nuclease P1 for 35 min at room temperature. Samples were then spotted onto a PEI-cellulose TLC plate and developed in 100 mM ammonium acetate, 5% acetic acid for 75 min. (B) PSTK assay using [14C]Ser. Phosphorimage of the separation of [14C]Ser-AMP and [14C]Sep-AMP on a PEI-cellulose TLC plate is shown. 2 µM tRNASec was incubated with 1.2 µM SerRS (lane 1) or 1.2 µM SerRS and 200 nM PSTK (lane 2) in the presence of 100 µM [14C]Ser for 45 min at 37°C. The aa-tRNASec products were purified as described in the Materials and Methods section, digested with nuclease P1 and chromatographed as above. (C) PSTK assay with γ-[32P]ATP as the phosphate donor. Phosphorimage of [32P]Sep-AMP on PEI-cellulose is shown. 1 µM Ser-tRNASer (lane 1) or 1 µM Ser-tRNASec (lane 2) was incubated with 100 nM PSTK with 1.67 µM γ-[32P]ATP for 45 min at 37°C. The reactions were purified over a Sephadex G25 Microspin column to remove unincorporated γ-[32P]ATP prior to digestion with nuclease P1 and TLC analysis as in (A).
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Figure 1: In vitro conversion of Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK. (A) PSTK assay with 32P-labeled tRNA. Representative phosphorimage of the separation of Ser-[32P]AMP, [32P]AMP and Sep-[32P]AMP by PEI cellulose chromatography is shown. 1 µM 32P-labeled tRNASec was incubated with 600 nM SerRS (lane 1) or 600 nM SerRS and 100 nM PSTK (lane 2) at 37°C for 45 min. Aliquots of the reactions were quenched with 100 mM sodium citrate, pH 5.0 and digested with 0.66 mg/ml nuclease P1 for 35 min at room temperature. Samples were then spotted onto a PEI-cellulose TLC plate and developed in 100 mM ammonium acetate, 5% acetic acid for 75 min. (B) PSTK assay using [14C]Ser. Phosphorimage of the separation of [14C]Ser-AMP and [14C]Sep-AMP on a PEI-cellulose TLC plate is shown. 2 µM tRNASec was incubated with 1.2 µM SerRS (lane 1) or 1.2 µM SerRS and 200 nM PSTK (lane 2) in the presence of 100 µM [14C]Ser for 45 min at 37°C. The aa-tRNASec products were purified as described in the Materials and Methods section, digested with nuclease P1 and chromatographed as above. (C) PSTK assay with γ-[32P]ATP as the phosphate donor. Phosphorimage of [32P]Sep-AMP on PEI-cellulose is shown. 1 µM Ser-tRNASer (lane 1) or 1 µM Ser-tRNASec (lane 2) was incubated with 100 nM PSTK with 1.67 µM γ-[32P]ATP for 45 min at 37°C. The reactions were purified over a Sephadex G25 Microspin column to remove unincorporated γ-[32P]ATP prior to digestion with nuclease P1 and TLC analysis as in (A).
Mentions: First, we 32P-labeled the terminal 3′-AMP of the tRNA using the exchange reaction of the E. coli CCA-adding enzyme in the presence of [α-32P]ATP. The radioactive tRNA was then serylated by pure M. maripaludis SerRS. After conversion of the Ser-tRNASec to Sep-tRNASec by M. jannaschii PSTK, the aa-tRNA products were digested by nuclease P1. The resulting mixture of Sep-[32P]AMP, [32P]AMP and Ser-[32P]AMP was separated on polyethyleneimine (PEI)-cellulose plates by TLC (Figure 1A), giving Rf values of 0.29, 0.50 and 0.85, respectively. This assay allows direct monitoring of the deacylation of the substrate Ser-tRNASec and the product Sep-tRNASec that inevitably occurs over the course of the reaction. We should note that in this study we used the transcript of the M. maripaludis tRNASec; while SerRS serylated both M. jannaschii Ser-tRNASec and M. maripaludis Ser-tRNASec to 70–80%, M. jannaschii Ser-tRNASec deacylated more rapidly than M. maripaludis Ser-tRNASec.Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Yale University, New Haven, CT 06520-8114, USA.

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 (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.

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Related in: MedlinePlus