Information on EC 6.5.1.6 - DNA ligase (ATP or NAD+)

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The expected taxonomic range for this enzyme is: Thermococcus

EC NUMBER
COMMENTARY hide
6.5.1.6
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RECOMMENDED NAME
GeneOntology No.
DNA ligase (ATP or NAD+)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(deoxyribonucleotide)n-3'-hydroxyl + 5'-(5'-diphosphoadenosine)-(deoxyribonucleotide)m = (deoxyribonucleotide)n+m + AMP
show the reaction diagram
(1c); catalytic reaction mechanism, detailed overview
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5'-adenosyl [DNA ligase]-Nepsilon-phosphono-L-lysine + 5'-phospho-(deoxyribonucleotide)m = 5'-(5'-diphosphoadenosine)-(deoxyribonucleotide)m + [DNA ligase]-L-lysine
show the reaction diagram
(1b); (2b)
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ATP + (deoxyribonucleotide)n-3'-hydroxyl + 5'-phospho-(deoxyribonucleotide)m = (deoxyribonucleotide)n+m + AMP + diphosphate
show the reaction diagram
ATP + [DNA ligase]-L-lysine = 5'-adenosyl [DNA ligase]-Nepsilon-phosphono-L-lysine + diphosphate
show the reaction diagram
(1a)
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NAD+ + (deoxyribonucleotide)n-3'-hydroxyl + 5'-phospho-(deoxyribonucleotide)m = (deoxyribonucleotide)n+m + AMP + beta-nicotinamide D-nucleotide
show the reaction diagram
NAD+ + [DNA ligase]-L-lysine = 5'-adenosyl [DNA ligase]-Nepsilon-phosphono-L-lysine + beta-nicotinamide D-nucleotide
show the reaction diagram
(2a)
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SYSTEMATIC NAME
IUBMB Comments
poly(deoxyribonucleotide)-3'-hydroxyl:5'-phospho-poly(deoxyribonucleotide) ligase (ATP or NAD+)
The enzymes from the archaea Thermococcus fumicolans and Thermococcus onnurineus show high activity with either ATP or NAD+, and significantly lower activity with TTP, GTP, and CTP. 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 or NAD+, 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. Different from EC 6.5.1.1, DNA ligase (ATP), EC 6.5.1.2, DNA ligase (NAD+) and EC 6.5.1.7, DNA ligase (ATP, ADP or GTP).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Q2Q452
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ADP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + phosphate + (deoxyribonucleotide)n+m
show the reaction diagram
Q2Q452
about 10% of the activity compared to ATP
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-
?
ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
GTP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
GMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
Q2Q452
about 20% of the activity compared to ATP
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-
?
NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m
AMP + beta-nicotinamide D-ribonucleotide + (deoxyribonucleotide)n+m
show the reaction diagram
TTP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
TMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
Q2Q452
about 20% of the activity compared to ATP
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-
?
UTP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
UMP + diphosphate + (deoxyribonucleotide)n+m
show the reaction diagram
Q2Q452
about 15% of the activity compared to ATP
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
the enzyme does not require monovalent cations to be active. 2 mM KCl or up to 5 mM of NaCl increase its activity up to 70%. Higher concentrations of these cations are inhibitory, but the enzyme still retains 50% activity with 100 mM NaCl or 100 mM KCl
Na+
the enzyme does not require monovalent cations to be active. 2 mM KCl or up to 5 mM of NaCl increase its activity up to 70%. Higher concentrations of these cations are inhibitory, but the enzyme still retains 50% activity with 100 mM NaCl or 100 mM KCl
Zn2+
Q2Q452
1 mM, 1.5 fold stimulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
Q2Q452
1 mM, about 25% loss of activity
Mg2+
while optimal activity is obtained with 2 mM, it is almost totally inhibited with 10 mM MgCl2
Mn2+
Q2Q452
1 mM, about 50% loss of activity
Ni2+
Q2Q452
1 mM, about 70% loss of activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.16
ATP
pH 6.5, 55°C
0.69
NAD+
pH 6.5, 55°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
Q2Q452
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
about 80% of maximal activity, pH 7.0: optimum, pH 7.5: about 10% of maximal activity
7 - 9
Q2Q452
pH 7.0: about 60% of maximal activity, pH 9.0: about 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 80
40°C: 50% of maximal activity, 80°C: about 50% of maximal activity
65 - 90
Q2Q452
65°C: about 50% of maximal activity, 90°C: about 95% of maximal activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
63000
Q2Q452
x * 63000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
Q2Q452
x * 63000, SDS-PAGE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
15 min, 50% loss of activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) pLysS