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T4 polynucleotide kinase (PNK) was originally discovered in the protein extracts of E. coli infected by T-even phage and has now become the main reagent used in molecular biology research. The product of the T4 early gene pseT, PNK catalyzes the transfer of γ-phosphate groups from adenosine triphosphate (ATP) or other nucleoside triphosphates to the 5'-hydroxyl groups of polynucleotides of different lengths, sequences and types. Both double-stranded and single-stranded DNA, RNA and individual 3'-phosphate nucleotide bases can be used as substrates for this enzyme. In addition, the enzyme has 5'-hydroxyl kinase and 2', 3'-cyclic phosphodiesterase activities against multiple substrates. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functional domains and forms a homotetramer. The C-terminal phosphate domain is homologous to the L-2-haloacid dehalogenase family, and the N-terminal domain is homologous to adenylate kinase.
T4 PNK is a homotetramer of 301 amino acid polypeptides. Although it has been widely used in nucleic acid biochemistry, the structure and mechanism of this bifunctional enzyme are still unknown. Early studies have shown that the C-terminus of PNK is involved in the 3'-phosphatase function, and the N-terminus is involved in the 5′-kinase reaction. Some studies have used site-directed mutagenesis to locate the basic composition of the active site of the 5'-kinase and the 3'-phosphatase activity. The amino acids required to catalyze the 5'-kinase reaction (Lys15, Ser16, Asp35, Arg38, Asp85 and Arg126) are mainly present at the N-terminus, while the residues necessary for 3'-phosphatase function (Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278) are mainly concentrated at the C-terminal. These results indicate that there is a domain boundary between Arg126 and Asp165. The C-terminal phosphatase domain can form a stable dimer, and the N-terminal domain is necessary for the enzyme to transition from dimer to tetramer. Studies have also shown that the 5'-kinase domains and 3'-phosphatase domains of PNK can be physically separated at the domain boundary, and the 5'-kinase activity may be affected by PNK oligomerization.
At present, the PNK N-terminal kinase domain PNK (1-181) has been resolved. This structure contains a continuous polypeptide from PNK residues 1 to 152, the N-terminal His-tag and the C-terminal fragment of amino acids 153 to 181 (the proximal fragments of the phosphatase domain) are disordered and have no electron density. The kinase domain consists of a central four-strand parallel β-sheet with three α-helices on each side. There is a Walker A-box motif (9GxxGxGKS16) between the first β-strand and the first α-helix. The classic P-loop structure that appears in many nucleotide-dependent phosphotransferases is found in the A-box. There are also two sulfate ions that bind to the P-loop and α2 in the PNK structure. The kinase monomer of the asymmetric unit is in contact with another monomer across a crystallographic 2-fold axis, and the α2–α3 loop and α1, β2, α2, α3 forms a contact surface of 2400 Å2 (1200 Å2 per protomer). The molecules at the interface are mainly hydrophobic, but also include hydrogen bonds between the main chain (from Asn31 amide to Ile41 carbonyl), from side chain to main chain (from Asn31 amide to Ser40 carbonyl) and from side chain to side chain side (Asp36 Oδ1 to His44Nε; Asp36 Oδ2 to His44Nε and Ser40 Oγ).
Fig 1. Structure of the 5′-kinase domain (Wang, L.K.; et al. 2002)
There are two ordered sulfate ions in the tertiary structure of PNK. The first sulfate has extensive contact with the backbone amide nitrogen of the P-loop and the Lys15 and Ser16 residues of the A-box, both Lys15 and Ser16 are essential for 5'-kinase activity. The network of sulfate and P-loop interactions in PNK crystals represents contacts made to the nucleotide β phosphate in the structures of numerous other P-loop-containing phosphotransferases. Therefore, it can be inferred that the sulfate defines the NTP binding site of PNK. The second sulfate ion in the tertiary structure corresponds to the phosphate acceptor site of other P-loop-containing phosphotransferases, it is 8.5Å away from the first sulfate ion. The second sulfate is coordinated with the main chain amide nitrogen of Thr86 and the side chain Oγ and the two terminal guanidinium nitrogens of Arg38(in α2).
Fig 2. PNK active site (Wang, L.K.; et al. 2002)
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