2ia5
T4 polynucleotide kinase/phosphatase with bound sulfate and magnesium.T4 polynucleotide kinase/phosphatase with bound sulfate and magnesium.
Structural highlights
FunctionKIPN_BPT4 Acts as a 5'-hydroxyl kinase, a 3'-phosphatase and a 2',3'-cyclic phosphodiesterase. Catalyzes the transfer of the terminal phosphate of ATP to the 5'-hydroxyl termini of ribo- and deoxyribonucleotides. In the presence of ADP the enzyme also catalyzes an exchange reaction. In the exchange reaction, an excess ADP causes the enzyme to transfer the 5' terminal phosphate from phosphorylated DNA to ADP. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedT4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity. Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase.,Zhu H, Smith P, Wang LK, Shuman S Virology. 2007 Sep 15;366(1):126-36. Epub 2007 May 9. PMID:17493655[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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