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Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

ABSTRACT: T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5'-kinase, but spared the 3'-phosphatase activity. Conservative substitutions of lysine or glutamine for Arg38 and Arg126 did not restore 5'-kinase activity. These results, together with previous mutational studies, highlight a constellation of five amino acids (Lys15, Ser16, Asp35, Arg38 and Arg126) that likely comprise the 5'-kinase active site. Four of these residues are conserved at the active sites of adenylate kinases (Adk), suggesting that Pnk and Adk are structurally and mechanistically related. We found that alanine substitutions for Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278 inactivated the 3'-phosphatase, but spared the 5'-kinase. Conservative substitutions of asparagine or glutamate for Asp165, Asp167 and Asp254 did not revive the 3'-phosphatase activity, nor did lysine substitutions for Arg176 and Arg213. Glutamate in lieu of Asp278 partially restored activity, whereas asparagine had no salutary effect. Alanine substitutions for Arg246 and Arg279 partially inactivated the 3'-phosphatase; the conservative R246K change restored activity, whereas R279K had no benefit. The essential phosphatase residues Asp165 and Asp167 are located within a 165DxDxT169 motif that defines a superfamily of phosphotransferases. Our data suggest that the 3'-phosphatase active site incorporates multiple additional functional groups.

SUBMITTER: Wang LK

PROVIDER: S-EPMC100346 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

Wang Li Kai LK Shuman Stewart S

Nucleic acids research 20020201 4

T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5'-kinase, but spared the 3'-phosphata ...[more]

PMID: 11842120

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Project description:T4 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.

Dataset Information

Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

Publications

Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

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