Literature summary for 3.1.99.B1 extracted from

Matsui, E.; Abe, J.; Yokoyama, H.; Matsui, I.
Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii (2004), J. Biol. Chem., 279, 16687-16696 .

Search for more literature for 3.1.99.B1

Cloned(Commentary)

Cloned (Comment)	Organism
expression of the mutant proteins in Escherichia coli BL21(DE3)	Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
F278A	the substitution of the aromatic residues with alanine leads to a large reduction in kcat value, although the mutants retains Km-value similar to that of the wild-type enzyme	Pyrococcus horikoshii
F278A/F279A	the kcat value of the mutant enzyme with the 5'-recess-end substrate and the the nick substrate is 10-20% of that of wild-type enzyme	Pyrococcus horikoshii
F278H/F279H	the Km value for the 5'-recess-end substrate is elevated 5times compared with that of wild-type enzyme, for the nick substrate the Km value is 60% that of wild-type enzyme	Pyrococcus horikoshii
F278L/F279L	the kcat value of the mutant enzyme with the 5'-recess-end substrate and the the nick substrate is 10-20% of that of wild-type enzyme	Pyrococcus horikoshii
F278L/F279L	the Km value for the 5'-recess-end substrate and the the nick substrate is lower than that of the wild-type enzyme	Pyrococcus horikoshii
F278W/F279W	the kcat value of the mutant enzyme with the 5'-recess-end substrate and the the nick substrate is 10-20% of that of wild-type enzyme	Pyrococcus horikoshii
F278W/F279W	the Km value for the 5'-recess-end substrate and the the nick substrate is lower than that of the wild-type enzyme	Pyrococcus horikoshii
F278Y/F279Y	the kcat value of the mutant enzyme with the 5'-recess-end substrate and the the nick substrate is around 70% of that of wild-type enzyme	Pyrococcus horikoshii
F278Y/F279Y	the Km value for the 5'-recess-end substrate and the the nick substrate is lower than that of the wild-type enzyme	Pyrococcus horikoshii
F279A	the substitution of the aromatic residues with alanine leads to a large reduction in kcat value, although the mutants retains Km-value similar to that of the wild-type enzyme	Pyrococcus horikoshii
F35A	the kcat and Km show no significant decrease compared with that of wild-type. For the nick substrate, the kcat value decreases 25fold compared with that of wild-type enzyme	Pyrococcus horikoshii
F35L	the kcat and Km show no significant decrease compared with that of wild-type	Pyrococcus horikoshii
F35Y	mutation causes an 17fold decrease and an 24fold decrease in kcat with the nick and 5'-recess-end substrates, respectively	Pyrococcus horikoshii
F79A	for the 5'-recess-end substrate, the kcat value decreases 71fold compared with that of wild-type enzyme. For the nick substrate, the kcat-value decreases 25fold compared with that of wild-type enzyme	Pyrococcus horikoshii
F79H	the kcat value of the mutant enzyme for the nick substrate is seven times lower than that of the wild-type enzyme. The Km value of the mutant enzyme for the 5'-recess-end substrate is 13fold higher than that of wild-type enzyme. The Km value of the mutant enzyme for the nick substrate is about 2 times higher than that of the wild-type enzyme	Pyrococcus horikoshii
F79L	for the 5'-recess-end substrate and for the the nick substrate, the kcat value is 20% of the wild-type value value	Pyrococcus horikoshii
F79W	the kcat value for the 5'-recess-end substrate and for the the nick substrate are almost the same as the wild-type values	Pyrococcus horikoshii
F79Y	the kcat value for the 5'-recess-end substrate and for the the nick substrate are almost the same as the wild-type values	Pyrococcus horikoshii
additional information	the aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contribute to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but play minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovers the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases	Pyrococcus horikoshii
Y33A	the substitution of the aromatic residues with alanine leads to a large reduction in kcat value (333fold decrease in exo-activity against the 5'-recess-end substrate), although the mutants retains Km-value similar to that of the wild-type enzyme. The exo-activity against the nick substrate, the kcat values decreases 53fold compared with that of wild-type enzyme	Pyrococcus horikoshii
Y33F	the kcat value for exo-activity against the 5'-recess-end substrate is about 25% compared to tht kcat value of wild-type enzyme, the Km value changes slightly compared with that of wild-type enzyme	Pyrococcus horikoshii
Y33H	the kcat value for exo-activity against the 5'-recess-end substrate is about 25% compared to tht kcat value of wild-type enzyme, the Km value changes slightly compared with that of wild-type enzyme	Pyrococcus horikoshii
Y33L	the muation leads to a large reduction in kcat value (1180fold decrease in exo-activity against the 5'-recess-end substrate) compared to tht kcat value of wild-type enzyme. The exo-activity against the nick substrate, the kcat values decreases 1343fold compared with that of wild-type enzyme	Pyrococcus horikoshii
Y33W	the kcat value for exo-activity against the 5'-recess-end substrate is about 25% compared to tht kcat value of wild-type enzyme, the Km value changes slightly compared with that of wild-type enzyme	Pyrococcus horikoshii

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O50123	-	-
Pyrococcus horikoshii OT-3	O50123	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
DNA + H2O	the aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contribute to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but play minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection)	Pyrococcus horikoshii	?	-	?
DNA + H2O	the aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contribute to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but play minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection)	Pyrococcus horikoshii OT-3	?	-	?
additional information	the enzyme possesses 5'-flap endonuclease and 5'->3' exonuclease activity	Pyrococcus horikoshii	?	-	?
additional information	the enzyme possesses 5'-flap endonuclease and 5'->3' exonuclease activity	Pyrococcus horikoshii OT-3	?	-	?

Synonyms

Synonyms	Comment	Organism
FEN-1	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	assay at	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
malfunction	the enzyme has important roles in DNA replication, repair, and recombination	Pyrococcus horikoshii