Information on EC 3.1.21.B2 - restriction endonuclease PabI

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The expected taxonomic range for this enzyme is: Pyrococcus abyssi

EC NUMBER
COMMENTARY hide
3.1.21.B2
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RECOMMENDED NAME
GeneOntology No.
restriction endonuclease PabI
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
the restriction enzyme cleaves 5'-GTAC and leaves 3'-TA overhangs (5'-GTA/C)
show the reaction diagram
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
DNA + H2O
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
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the enzyme is active in NaCl concentrations ranging from 100 to 200 mM
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
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active up to
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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the enzyme forms a tetrameric structure to sandwich the double-stranded DNA and the tetrameric structure is stabilized by four salt bridges
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallization experiments with the mutant enzyme R32A/E63A-nonspecific dsDNA complex are performed using the sitting-drop vapour-diffusion method at 20°C. The crystals of the mutant enzyme R32A/E63A-nonspecific dsDNA complex are obtained using a reservoir solution of 0.2 M calcium acetate, 0.1 M imidazole (pH 8.0), and 10% PEG 8000.The structure of enzyme in complex with double-stranded DNA without the R.PabI recognition sequence is determined by X-ray crystallography. The 1.9 A resolution structure of the complex shows that the enzyme forms a tetrameric structure to sandwich the double-stranded DNA and the tetrameric structure is stabilized by four salt bridges
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R70D
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the mutation does not show an effect on the nonspecific dsDNA binding affinity. The mutations would prevent the tetramerization of the enzyme on nonspecific dsDNA due to the electrostatic repression of their side chains
Y68F
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the mutant enzyme possesses approximately the same base-specific DNA binding ability as the wild-type enzyme but has reduced DNA glycosylase activity. The mutations would prevent the tetramerization of the enzyme on nonspecific dsDNA due to the electrostatic repression of their side chains
Y68F/D71R
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the wild-type enzyme forms a tetramer with nonspecific dsDNA and the tetramerization is prevented by the D71R mutation. The mutations would prevent the tetramerization of the enzyme on nonspecific dsDNA due to the electrostatic repression of their side chains. The Y68F/D71R mutant would also prevent the tetramerization of the enzyme on nonspecific dsDNA due to the steric hindrance of the long side chain of D71R. The DNA glycosylase activity with the 24 bp dsDNA substrate is 74% compared with the mutant enzyme Y68Y (a mutant enzyme that possesses approximately the same base-specific DNA binding ability as the wild-type enzyme but has reduced DNA glycosylase activity). The activity with the 500 bp dsDNA substrate is 34% compared with the mutant enzyme Y68Y. The activity with the 3000 bp dsDNA substrate is 19% compared with the mutant enzyme Y68Y
Y68F/R26A
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the DNA glycosylase activity with the 24 bp dsDNA substrate is 65% compared with the mutant enzyme Y68Y (a mutant enzyme that possesses approximately the same base-specific DNA binding ability as the wild-type enzyme but has reduced DNA glycosylase activity). The activity with the 500 bp dsDNA substrate is 55% compared with the mutant enzyme Y68Y. The activity with the 3000 bp dsDNA substrate is 27% compared with the mutant enzyme Y68Y
Y68F/R70D
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the DNA glycosylase activity with the 24 bp dsDNA substrate is 127% compared with the mutant enzyme Y68Y (a mutant enzyme that possesses approximately the same base-specific DNA binding ability as the wild-type enzyme but has reduced DNA glycosylase activity). The activity with the 500 bp dsDNA substrate is 76% compared with the mutant enzyme Y68Y. The activity with the 3000 bp dsDNA substrate is 19% compared with the mutant enzyme Y68Y
Y68F/R70D/D71R
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the DNA glycosylase activity with the 24 bp dsDNA substrate is 105% compared with the mutant enzyme Y68Y (a mutant enzyme that possesses approximately the same base-specific DNA binding ability as the wild-type enzyme but has reduced DNA glycosylase activity). The activity with the 500 bp dsDNA substrate is 71% compared with the mutant enzyme Y68Y. The activity with the 3000 bp dsDNA substrate is 40% compared with the mutant enzyme Y68Y