Polynucleotide 5'-hydroxyl-kinase

 

This enzyme catalyses 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities. These activities modify the ends of nicked tRNA generated by bacterial response to infection. These catalytic activities are dependent on magnesium as a cofactor. The kinase activity resides in an N-terminal domain, whilst the phosphatase activity resides in the C-terminal domain.
T4 PNK also has the ability to overcome some bacterial suicide defence mechanisms.
It is a member of a family of 5'-kinase/3'-phosphatases that mend broken strands in nucleic acids in conjunction with a RNA/DNA ligase. The kinase domain belongs to the adenylate kinase family whilst the phosphatase domain belongs to the L-2-haloacid dehalogenase.

 

Reference Protein and Structure

Sequence
P06855 UniProt (2.7.1.78, 3.1.3.34) IPR027417 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T4 (Virus) Uniprot
PDB
1ltq - CRYSTAL STRUCTURE OF T4 POLYNUCLEOTIDE KINASE (2.33 Å) PDBe PDBsum 1ltq
Catalytic CATH Domains
3.40.50.1000 CATHdb 3.40.50.300 CATHdb (see all for 1ltq)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.1.78)

ATP(4-)
CHEBI:30616ChEBI
+
5'-end 2'-deoxyribonucleoside residue
CHEBI:136416ChEBI
→
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
+
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
Alternative enzyme names: 5'-hydroxyl RNA kinase, 5'-hydroxyl polynucleotide kinase, 5'-hydroxyl polyribonucleotide kinase, ATP:5'-dephosphopolynucleotide 5'-phosphatase, DNA 5'-hydroxyl kinase, DNA kinase, PNK, Polynucleotide 5'-hydroxyl kinase (phosphorylating), Polynucleotide kinase, Polynucleotide 5'-hydroxy-kinase,

Enzyme Mechanism

Introduction

PNK catalyses the transfer of the gamma-phosphate from adenosine triphosphate or other nucleoside triphosphates to the 5'-OH of polynucleotides. The activity proceeds via an ordered sequential mechanism. Phosphoryl transfer results in an inversion of configuration at the phosphorous. The attacking nucleophile is the 5'-OH of the nucleoside, attacking the gamma phosphorous of ATP. Abstraction of the 5'-OH proton by Asp 35 activates the nucleophile, giving it the role of general base catalyst. The pentacoordinate transition state is stabilised by Lys 15 and Arg 126 side chains. The phosphatase proceeds via a covalent phospho-aspartic acid and is dependent on magnesium for activity. Asp 165 acts as the nucleophile attacking the 3'-phosphorous in an SN2 phosphoryl transfer reaction.

Catalytic Residues Roles

UniProt PDB* (1ltq)
Lys15 Lys15A Stabilises the pentacoordinate transition state in the kinase reaction. electrostatic stabiliser
Arg126 Arg126A Stabilises the pentacoordinate transition state in the kinase. electrostatic stabiliser
Asp35 Asp35A General base catalyst that activates the 5'-OH to increase its nucleophilicity, in the kinase domain. proton shuttle (general acid/base)
Asp165 Asp165A Nucleophile in the phosphatase, attacks the 3'-phosphorous in an SN2 phosphoryl transfer reaction. covalent catalysis
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Wang LK et al. (2002), EMBO J, 21, 3873-3880. Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme. DOI:10.1093/emboj/cdf397. PMID:12110598.
  2. Das U et al. (2013), Biochemistry, 52, 4734-4743. Structural and biochemical analysis of the phosphate donor specificity of the polynucleotide kinase component of the bacterial pnkp•hen1 RNA repair system. DOI:10.1021/bi400412x. PMID:23721485.
  3. Galburt EA et al. (2002), Structure, 10, 1249-1260. Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase. PMID:12220496.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys15A electrostatic stabiliser
Arg126A electrostatic stabiliser
Asp35A proton shuttle (general acid/base)
Asp165A covalent catalysis

Chemical Components

Step 1.

Contributors

Hannah Gilbert, Gary McDowell, Gemma L. Holliday, Morwenna Hall