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Information on EC 6.5.1.1 - DNA ligase (ATP) and Organism(s) Pyrococcus furiosus and UniProt Accession P56709

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EC Tree
IUBMB Comments
The enzyme catalyses the ligation of DNA strands with 3'-hydroxyl and 5'-phosphate termini, forming a phosphodiester and sealing certain types of single-strand breaks in duplex DNA. Catalysis occurs by a three-step mechanism, starting with the activation of the enzyme by ATP, forming a phosphoramide bond between adenylate and a lysine residue. The adenylate group is then transferred to the 5'-phosphate terminus of the substrate, forming the capped structure 5'-(5'-diphosphoadenosine)-[DNA]. Finally, the enzyme catalyses a nucleophilic attack of the 3'-OH terminus on the capped terminus, which results in formation of the phosphodiester bond and release of the adenylate. RNA can also act as substrate, to some extent. cf. EC 6.5.1.2, DNA ligase (NAD+), EC 6.5.1.6, DNA ligase (ATP or NAD+), and EC 6.5.1.7, DNA ligase (ATP, ADP or GTP).
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Pyrococcus furiosus
UNIPROT: P56709
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The taxonomic range for the selected organisms is: Pyrococcus furiosus
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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ATP
+
(deoxyribonucleotide)n-3'-hydroxyl
+
5'-phospho-(deoxyribonucleotide)m
=
(deoxyribonucleotide)n+m
+
AMP
+
diphosphate
+
+
5'-phospho-(deoxyribonucleotide)m
=
+
+
Synonyms
dna ligase, dna repair enzyme, dna ligase i, t4 dna ligase, dna ligase iv, dna ligase iii, ligase 1, dna ligase ii, polynucleotide ligase, dna ligase 1, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PF1635
locus name
Deoxyribonucleate ligase
-
-
-
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Deoxyribonucleic acid joinase
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-
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Deoxyribonucleic acid ligase
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Deoxyribonucleic acid repair enzyme
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Deoxyribonucleic acid-joining enzyme
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-
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Deoxyribonucleic joinase
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Deoxyribonucleic ligase
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Deoxyribonucleic repair enzyme
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-
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Deoxyribonucleic-joining enzyme
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DNA joinase
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DNA ligase
DNA ligase I
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DNA ligase II
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-
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DNA ligase III
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-
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DNA ligase IV homolog
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-
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DNA repair enzyme
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-
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DNA-joining enzyme
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Lig(Tk)
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-
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Pfu DNA ligase
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-
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Polydeoxyribonucleotide synthase (ATP)
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Polydeoxyribonucleotide synthase [ATP]
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Polynucleotide ligase
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Sealase
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SYSTEMATIC NAME
IUBMB Comments
poly(deoxyribonucleotide)-3'-hydroxyl:5'-phospho-poly(deoxyribonucleotide) ligase (ATP)
The enzyme catalyses the ligation of DNA strands with 3'-hydroxyl and 5'-phosphate termini, forming a phosphodiester and sealing certain types of single-strand breaks in duplex DNA. Catalysis occurs by a three-step mechanism, starting with the activation of the enzyme by ATP, forming a phosphoramide bond between adenylate and a lysine residue. The adenylate group is then transferred to the 5'-phosphate terminus of the substrate, forming the capped structure 5'-(5'-diphosphoadenosine)-[DNA]. Finally, the enzyme catalyses a nucleophilic attack of the 3'-OH terminus on the capped terminus, which results in formation of the phosphodiester bond and release of the adenylate. RNA can also act as substrate, to some extent. cf. EC 6.5.1.2, DNA ligase (NAD+), EC 6.5.1.6, DNA ligase (ATP or NAD+), and EC 6.5.1.7, DNA ligase (ATP, ADP or GTP).
CAS REGISTRY NUMBER
COMMENTARY hide
9015-85-4
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
ATP + ADP
P1-(5'-adenosyl),P3-(5'-adenosyl)triphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + CDP
P1-(5'-adenosyl),P3-(5'-cytidyl)triphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + CTP
P1-(5'-adenosyl),P4-(5'-cytidyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + dCTP
P1-(5'-adenosyl),P4-[5'-(2'-deoxycytidyl)]tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + dGTP
P1-(5'-adenosyl),P4-[5'-(2'-deoxyguanosyl)]tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + dTTP
P1-(5'-adenosyl),P4-(5'-thymidyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + GDP
P1-(5'-adenosyl),P3-(5'-guanosyl)triphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + GTP
P1-(5'-adenosyl),P4-(5'-guanosyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + UTP
P1-(5'-adenosyl),P4-(5'-uridinyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + XDP
P1-(5'-adenosyl),P4-(5'-xanthosyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + XTP
P1-(5'-adenosyl),P4-(5'-xanthosyl)tetraphosphate + diphosphate
show the reaction diagram
-
-
-
?
ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + diphosphate + (deoxyribonucleotide)m+n
show the reaction diagram
-
-
-
-
?
additional information
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
optimal concentration: 4 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
proliferating cell nuclear antigen
stimulatory effect of Pyrococcus furiosus proliferating cell nuclear antigen (PCNA) on the enzyme activity of Pyrococcus furiosus DNA ligase is observed not at low ionic strength, but at a high salt concentration, at which a DNA ligase alone cannot bind to a nicked DNA substrate. Identification of the amino acid residues that are critical for PCNA binding in a loop structure located in the N-terminal DNA-binding domain of Ppyrococcus furiosus DNA ligase. The pentapeptide motif QKSFF is involved in the PCNA-interacting motifs, in which Gln and the first Phe are especially important for stable binding with PCNA
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.001
ATP
pH 7.5, 70°C, synthesis of P1-(5'-adenosyl),P4-(5'-guanosyl)tetraphosphate
0.4
GTP
pH 7.5, 70°C, synthesis of P1-(5'-adenosyl),P4-(5'-guanosyl)tetraphosphate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
1.8 A resolution structure of Pyrococcus furiosus DNA ligase. The enzyme comprises the N-terminal DNA binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes
hanging-drop vapour diffusion method, crystals of the archaeal DNA ligase from Pyrococcus furiosus aree obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 A resolution. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 A, beta = 108.9°. The asymmetric unit contains one ligase molecule
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in complex with the AMP-DNA reaction intermediate
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D540R/delC4
DelC4 i.e. deletion of the four C-terminal residues, nick-joining activities of the mutant is enhanced as compared to that of the D540R single substitution
D540R/DELTAC4
the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with adenylylation domain, effectively enhances the activity
D540R/K554A/K558A
K249A
mutant enzyme shows no no adenylyltransferase activity
K534A
the wild-type R531A and mutant K534A enzymes exhibit almost the same DNA ligation activities both in the presence and in the absence of externally added ATP, contains AMP in the crystal
molecular biology
the enzyme is used in the ligase chain reaction
R531A
the wild-type R531A and mutant K534A enzymes exhibit almost the same DNA ligation activities both in the presence and in the absence of externally added ATP, contains AMP in the crystal
R531A/K534A
the mutations of both basic residues (R531A and K534A) severely affected the ligation activity, especially in the absence of ATP, does not contain AMP in the crystal
D540A
-
mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme, the mutant enzyme exhibits activity about twice as high as that of the wild type within 10 min
D540A/Q547A/K554A/K558A
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the mutant enzyme exhibits activity about twice as high as that of the wild type within 10 min. The D540A ligation is almost the same as that of the D540A/Q547A/K554A/K558A mutant enzyme, thus implying that a single substitution for Asp540 might exert a more dominant effect than the substitutions of the other three polar and ionic residues at the C terminus
D540K
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
D540R
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
D540S
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
Q547A/K554A/K558A
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nick ligation activity of the mutant is slightly higher than that of the wild type enzyme
R544A
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mutant R544A displays a notable reduction in nick-joining activity (less than 45% of the input substrate ligated) in comparison with that of mutant R544A/Q547A/K554A/K558A
R544A/Q547A/K554A/K558A
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mutant enzyme exhibits low activity. Mutant R544A displays a notable reduction in nick-joining activity (less than 45% of the input substrate ligated) in comparison with that of mutant R544A/Q547A/K554A/K558A
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
overexpression of wild-type and mutant enzymes in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pascal, J.M.
DNA and RNA ligases: structural variations and shared mechanisms
Curr. Opin. Struct. Biol.
18
96-105
2008
Homo sapiens, Pyrococcus furiosus, Saccharolobus solfataricus
Manually annotated by BRENDA team
Nishida, H.; Tsuchiya, D.; Ishino, Y.; Morikawa, K.
Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus
Acta Crystallogr. Sect. F
61
1100-1102
2005
Pyrococcus furiosus
Manually annotated by BRENDA team
Sillero, M.A.G.; Montes, M.; de Diego, A.; del Valle, M.; Atencia, E.A.; Sillero, A.
Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates
Extremophiles
6
45-50
2002
Pyrococcus furiosus (P56709), Pyrococcus furiosus
Manually annotated by BRENDA team
Kiyonari, S.; Takayama, K.; Nishida, H.; Ishino, Y.
Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen
J. Biol. Chem.
281
28023-28032
2006
Pyrococcus furiosus (P56709), Pyrococcus furiosus
Manually annotated by BRENDA team
Nishida, H.; Kiyonari, S.; Ishino, Y.; Morikawa, K.
The closed structure of an archaeal DNA ligase from Pyrococcus furiosus
J. Mol. Biol.
360
956-967
2006
Pyrococcus furiosus (P56709), Pyrococcus furiosus
Manually annotated by BRENDA team
Tanabe, M.; Ishino, S.; Ishino, Y.; Nishida, H.
Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity
FEBS Lett.
588
230-235
2014
Pyrococcus furiosus (P56709), Pyrococcus furiosus
Manually annotated by BRENDA team
Tanabe, M.; Ishino, S.; Yohda, M.; Morikawa, K.; Ishino, Y.; Nishida, H.
Structure-based mutational study of an archaeal DNA ligase towards improvement of ligation activity
ChemBioChem
13
2575-2582
2012
Pyrococcus furiosus, Thermococcus sp.
Manually annotated by BRENDA team