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Information on EC 2.7.1.78 - polynucleotide 5'-hydroxyl-kinase
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2.7.1.78 polynucleotide 5'-hydroxyl-kinaseIUBMB Comments
Also acts on 5'-dephospho-RNA 3'-mononucleotides.
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Word Map
- 2.7.1.78
- termini
- strand
-
single-stranded
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gamma-32patp
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exonuclease
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32p-postlabeling
-
5\'-hydroxyl
- bacteriophage
- phosphodiesterase
-
3'-phosphatase
- nick
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32p-labeled
- duplex
- 3'-monophosphate
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5'-phosphorylated
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5'-terminal
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postlabeling
- dsdna
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anticodon
-
5\'-termini
-
klenow
- xrcc4
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dinucleoside
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micrococcal
- 3',5'-bisphosphate
- synthesis
- primase
-
carcinogen-dna
-
phosphotriester
-
t4-infected
- oligoribonucleotide
- analysis
- biotechnology
- medicine
The enzyme appears in viruses and cellular organisms
Synonyms
pnk, polynucleotide kinase, t4 polynucleotide kinase, t4 pnk, hd-pnk, dna kinase, hpnkp, 5'-oh polynucleotide kinase, 5'-hydroxyl polynucleotide kinase, phoclp1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PNKP
5'-hydroxyl polynucleotide kinase
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5'-hydroxyl polyribonucleotide kinase
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5'-hydroxyl RNA kinase
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ATP:5'-dephosphopolynucleotide 5'-phosphatase
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DNA 5'-hydroxyl kinase
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DNA kinase
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kinase (phosphorylating), polynucleotide 5'-hydroxyl
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PNK
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polynucleotide 5'-hydroxyl kinase (phosphorylating)
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polynucleotide 5'-hydroxyl-kinase
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polynucleotide kinase
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PNKP
the enzyme is composed of three catalytic modules: an N-terminal 5'-OH polynucleotide kinase, a central 2',3' phosphatase, and a C-terminal ligase polynucleotide kinase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + 5'-dephospho-DNA = ADP + 5'-phospho-DNA
Asp38, as general base, activates the polynucleotide 5'-OH for its nucleophilic attack on the gamma phosphorus and Lys21 and Mg2+ stabilize the transition state, catalytic mechanism, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phospho group transfer
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-
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SYSTEMATIC NAME
IUBMB Comments
ATP:5'-dephosphopolynucleotide 5'-phosphotransferase
Also acts on 5'-dephospho-RNA 3'-mononucleotides.
CAS REGISTRY NUMBER
COMMENTARY
37211-65-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + 5'-dephospho-DNA
ADP + 5'-phospho-DNA
substrate is a synthetic 36-mer 5'-OH DNA oligonucleotide d(CCTGTTCTTATTGGCCTCCTGGCATACCTTTTCCGG)
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-
?
ATP + 5'-dephospho-DNA
ADP + 5'-phospho-DNA
the enzyme makes hydrogen bonds to the ribose 2'-hydroxyls of the 5'-terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83
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?
additional information
?
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Pnkp is a bifunctional enzyme: 2,3' cyclic phosphate and 5-OH ends are substrates for healing and sealing by Pnkp and Hen1, The 5' end is phosphorylated by the Pnkp kinase and the 2',3' cyclic phosphate is removed by the Pnkp phosphatase
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?
additional information
?
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Pnkp is a bifunctional enzyme: 2,3' cyclic phosphate and 5-OH ends are substrates for healing and sealing by Pnkp and Hen1, The 5' end is phosphorylated by the Pnkp kinase and the 2',3' cyclic phosphate is removed by the Pnkp phosphatase
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-
?
additional information
?
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the alpha and beta phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides, the gamma phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg2+ bridge the ATP beta and gamma phosphates
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?
additional information
?
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the alpha and beta phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides, the gamma phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg2+ bridge the ATP beta and gamma phosphates
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + 5'-dephospho-DNA
ADP + 5'-phospho-DNA
the enzyme makes hydrogen bonds to the ribose 2'-hydroxyls of the 5'-terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83
-
-
?
additional information
?
-
Pnkp is a bifunctional enzyme: 2,3' cyclic phosphate and 5-OH ends are substrates for healing and sealing by Pnkp and Hen1, The 5' end is phosphorylated by the Pnkp kinase and the 2',3' cyclic phosphate is removed by the Pnkp phosphatase
-
-
?
additional information
?
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Pnkp is a bifunctional enzyme: 2,3' cyclic phosphate and 5-OH ends are substrates for healing and sealing by Pnkp and Hen1, The 5' end is phosphorylated by the Pnkp kinase and the 2',3' cyclic phosphate is removed by the Pnkp phosphatase
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
ATP is bound within a crescent-shaped groove formed by the P-loop (15GSSGSGKST23) and an overlying helix-loop-helix lid
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
required, the P-loop lysine (Lys21) and the catalytic Mg2+ bridge the ATP beta and gamma phosphates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0028
5'-HO-CCGACCAACGAAGGT
wild type enzyme, at pH 7.0 and 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.072
5'-HO-CCGACCAACGAAGGT
wild type enzyme, at pH 7.0 and 37°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
the enzyme is part of the Pnkp-Hen1 RNA repair pathway, overview
additional information
additional information
structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system, the enzyme has an autonomous kinase domain, overview. Pnkp-Hen1 RNA repair pathway confers protective immunity to recurrent RNA damage
additional information
-
structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system, the enzyme has an autonomous kinase domain, overview. Pnkp-Hen1 RNA repair pathway confers protective immunity to recurrent RNA damage
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). A3DJ38_ACET2
Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372)
870
0
98776
TrEMBL
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PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
additional information
additional information
the protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain
additional information
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the protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complexes with GTP, CTP, UTP and dATP, hanging drop vapor diffusion method, using 100 mM sodium citrate, pH 5.6, 100 mM MgCl2, 16-18% (w/v) PEG-3350
kinase domain of enzyme Pnkp bound to ATP-Mg2+ or ADP-Mg2+, by hanging drop vapor diffusion, protein solution containing 0.6 mM kinase, 2 mM ATP, and 10 mM MgCl2 with an equal volume of precipitant solution containing 100 mM HEPES, pH 7.5, 200 mM MgCl2, and 30% PEG 400, 22°C, 2 days, X-ray diffraction structure determination and analysis at 2.1 A resolution
mutant enzyme D38N in complex with GTP-Mg2+ and a 5'-OH RNA oligonucleotide, hanging drop vapor diffusion method, using 100 mM sodium citrate (pH 5.0), 100 mM MgCl2, 18-24% (w/v) PEG-6000
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D38E
site-directed mutagenesis, the mutant shows 6% of wild-type activity
K21Q
site-directed mutagenesis, the mutant shows 100fold reduced activity compared to the wild-type enzyme
K21R
site-directed mutagenesis, the mutant shows 15fold reduced activity compared to the wild-type enzyme
K95A
site-directed mutagenesis, inactive mutant, the phenotype is benign
Q51A
the mutation reduces kinase specific activity about 3fold
R116A
the mutation elicits a 10fold increase in Km for ATP, but has little effect on the turnover number value leading to 83% of wild type activity
R41K
site-directed mutagenesis, the mutant shows about 65% reduced activity compared to the wild-type enzyme
R41Q
site-directed mutagenesis, the mutant shows 100fold reduced activity compared to the wild-type enzyme
S22T
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
T54A
the mutation reduces kinase specific activity about 3fold
V129A
the mutation reduces kinase specific activity about 3fold
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-agarose column chromatography and Superdex 200 gel filtration
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Wang, L.K.; Das, U.; Smith, P.; Shuman, S.
Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system
RNA
18
2277-2286
2012
Acetivibrio thermocellus (A3DJ38), Acetivibrio thermocellus
Das, U.; Wang, L.K.; Smith, P.; Shuman, S.
Structural and biochemical analysis of the phosphate donor specificity of the polynucleotide kinase component of the bacterial pnkp-hen1 RNA repair system
Biochemistry
52
4734-4743
2013
Acetivibrio thermocellus (A3DJ38), Acetivibrio thermocellus
Das, U.; Wang, L.K.; Smith, P.; Munir, A.; Shuman, S.
Structures of bacterial polynucleotide kinase in a Michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5-phosphorylated oligonucleotide
J. Bacteriol.
196
4285-4292
2014
Acetivibrio thermocellus (A3DJ38), Acetivibrio thermocellus, Acetivibrio thermocellus DSM 1237 (A3DJ38)
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