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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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Phylogenetic tree of the kinase domains of PSTK, Kti12 and representative groups of closely related ‘DxTN’ kinases, which include cellular and viral polynucleotide kinases (Pnk) and the phosphoserine/phosphothreonine kinases (PrpA). Organism names are color-coded: Archaea (blue), Eukarya (green), Bacteria (red) and viruses (purple). Bootstrap values for major branches are shown. All bootstrap values are included in Supplementary Figure 3.
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Figure 9: Phylogenetic tree of the kinase domains of PSTK, Kti12 and representative groups of closely related ‘DxTN’ kinases, which include cellular and viral polynucleotide kinases (Pnk) and the phosphoserine/phosphothreonine kinases (PrpA). Organism names are color-coded: Archaea (blue), Eukarya (green), Bacteria (red) and viruses (purple). Bootstrap values for major branches are shown. All bootstrap values are included in Supplementary Figure 3.

Mentions: The strong similarity of PSTK and Kti12 throughout the full-length of the molecule along with misannotations in the sequence databases led to a previous misclassification of some Kti12 proteins as PSTKs (4). In addition to presenting a resolved phylogeny of PSTK and Kti12 (Supplementary Data Figures 1 and 2), we also computed the phylogenetic root between these proteins. Such a root helps determine if Kti12 is derived from the eukaryotic PSTK or if Kti12 diverged from PSTK at some earlier time. A set of more distantly related (so-called out group) sequences is required to place the root. Since PSTK and Kti12 only display homology with other proteins in their kinase domain, their root must be determined from a phylogeny based only on the kinase domain (Figure 9). This tree shows a deep separation between the PSTK/Kti12 cluster and the other kinases in their family.Figure 9.


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

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

Phylogenetic tree of the kinase domains of PSTK, Kti12 and representative groups of closely related ‘DxTN’ kinases, which include cellular and viral polynucleotide kinases (Pnk) and the phosphoserine/phosphothreonine kinases (PrpA). Organism names are color-coded: Archaea (blue), Eukarya (green), Bacteria (red) and viruses (purple). Bootstrap values for major branches are shown. All bootstrap values are included in Supplementary Figure 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275090&req=5

Figure 9: Phylogenetic tree of the kinase domains of PSTK, Kti12 and representative groups of closely related ‘DxTN’ kinases, which include cellular and viral polynucleotide kinases (Pnk) and the phosphoserine/phosphothreonine kinases (PrpA). Organism names are color-coded: Archaea (blue), Eukarya (green), Bacteria (red) and viruses (purple). Bootstrap values for major branches are shown. All bootstrap values are included in Supplementary Figure 3.
Mentions: The strong similarity of PSTK and Kti12 throughout the full-length of the molecule along with misannotations in the sequence databases led to a previous misclassification of some Kti12 proteins as PSTKs (4). In addition to presenting a resolved phylogeny of PSTK and Kti12 (Supplementary Data Figures 1 and 2), we also computed the phylogenetic root between these proteins. Such a root helps determine if Kti12 is derived from the eukaryotic PSTK or if Kti12 diverged from PSTK at some earlier time. A set of more distantly related (so-called out group) sequences is required to place the root. Since PSTK and Kti12 only display homology with other proteins in their kinase domain, their root must be determined from a phylogeny based only on the kinase domain (Figure 9). This tree shows a deep separation between the PSTK/Kti12 cluster and the other kinases in their family.Figure 9.

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.

Show MeSH
Related in: MedlinePlus