Literature summary for 4.2.99.18 extracted from

Castillo-Acosta, V.M.; Ruiz-Perez, L.M.; Yang, W.; Gonzalez-Pacanowska, D.; Vidal, A.E.
Identification of a residue critical for the excision of 3-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family (2009), Nucleic Acids Res., 37, 1829-1842.

Search for more literature for 4.2.99.18

Cloned(Commentary)

Cloned (Comment)	Organism
Generation of random mutations in the ape1 gene and selection of variants that confer protection against H2O2. Random library of ape1 mutants is generated by transforming the mutator strain XL1-Red with the prokaryotic ape1 plasmid pKK-ape1. The H2O2-resistant phenotype (mutant D70A) is expressed in Escherichia coli strain BW528 (xth nfo), which is deficient in the major AP endonucleases, exonuclease III and endonuclease IV	Homo sapiens
mutant A138D is produced by site-directed mutagenesis and expressed in Escherichia coli strain BW528. Overexpression of LMAPA138D sensitizes Leishmania cells to oxidative agents.	Leishmania major

Cloned (Comment)

Organism

Generation of random mutations in the ape1 gene and selection of variants that confer protection against H2O2. Random library of ape1 mutants is generated by transforming the mutator strain XL1-Red with the prokaryotic ape1 plasmid pKK-ape1. The H2O2-resistant phenotype (mutant D70A) is expressed in Escherichia coli strain BW528 (xth nfo), which is deficient in the major AP endonucleases, exonuclease III and endonuclease IV

Homo sapiens

mutant A138D is produced by site-directed mutagenesis and expressed in Escherichia coli strain BW528. Overexpression of LMAPA138D sensitizes Leishmania cells to oxidative agents.

Leishmania major

Protein Variants

Protein Variants	Comment	Organism
A138D	production by site-directed mutagenesis	Leishmania major
D70A	mutant increases capacity to remove 3'-blocking ends in vitro	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
Mg2+	In case of APE 1, the excision being severely inhibited (below 25%) at 10 mM and higher ion concentrations. APE1 mutant D70A is more refractory to Mg2+ inhibition, thus still retaining about 50% of activity at 20 mM Mg2+. In case of a THF-containing oligonucleotide as substrate, inhibition is not evidenced until 80mM is reached.	Homo sapiens
Mg2+	In case of LMAP mutant A138D, the excision being severely inhibited (below 25%) at 10 mM and higher ion concentrations. Wild-type enzyme is more refractory to Mg2+ inhibition, thus still retaining about 50% of activity at 20 mM Mg2+	Leishmania major

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.000136	-	THF-containing oligonucleotide	APE1 mutant D70A, AP endonuclease activity	Homo sapiens
0.000154	-	THF-containing oligonucleotide	Ape1, AP endonuclease activity	Homo sapiens
0.000184	-	THF-containing oligonucleotide	LMAP mutant A138D, AP endonuclease activity	Leishmania major
0.0002	-	THF-containing oligonucleotide	LMAP, AP endonuclease activity	Leishmania major
0.000884	-	DNA	LMAP mutant A138D, 3'-phosphodiesterase activity	Leishmania major
0.001365	-	DNA	LMAP, 3'-phosphodiesterase activity	Leishmania major
0.001587	-	DNA	APE1 mutant D70A, 3'-phosphodiesterase activity	Homo sapiens
0.001689	-	DNA	APE1, 3'-phosphodiesterase activity	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	APE1 reaches a maximum of 3'-phosphoglycolate excision activity at Mg2+ concentrations around 2.5 mM, APE1 mutant D70A reaches maximum activity at higher metal concentrations. In case of a THF-containing oligonucleotide as substrate, APE1 exhibits the highest AP endonuclease activity at 5 mM of metal while the specific activity of APE1 mutant D70A did not reach a maximum until 40 mM of Mg2+ is added to the reaction.	Homo sapiens
Mg2+	LMAP mutant A138D reaches a maximum of 3'-PG excision activity at Mg2+ concentrations around 2.5 mM, wild-type enzyme reaches maximum activity at higher metal concentrations. In case of a THF-containing oligonucleotide as substrate, the parasite enzymes LMAP and LMAPA138D, obtains the activity peaks at different magnesium concentrations (10 and 2.5 mM).	Leishmania major

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
DNA	Leishmania major	DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase, apurinic, apyrimidinic (AP) lyases	fragments of DNA	-	?
DNA	Homo sapiens	DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases	fragments of DNA	-	?
additional information	Homo sapiens	APE1 can incise DNA at the 5'-position of oxidized pyrimidine bases such as 5,6-dihydro-thymine or 5,6-dihydro-2'-deoxyuracil (DHU), thus initiating a repair process known as nucleotide incision repair (NIR)	?	-	?
additional information	Homo sapiens	APE1 has DNA 3'-phosphatase activity in vitro and 3' to 5' exonuclease activity, which could be physiologically relevant in the removal of mismatched or damaged nucleotides incorporated during the synthesis step of BER	?	-	?
additional information	Homo sapiens	In addition to the exonuclease function, human APE1 is endowed with another enzymatic activity potentially relevant for the protection against oxidative damage	?	-	?
additional information	Homo sapiens	In human cells, APE1 excises sugar fragments that block the 3'-ends thus facilitating DNA repair synthesis	?	-	?
additional information	Leishmania major	LMAP shares with APE1 the overall 3D structure and most of the catalytic groups in their active sites and can catalyze the removal of damaged nucleotides and phosphate groups from 3'-ends more efficiently than APE1	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-
Leishmania major	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
histidine-tagged AP endonucleases are purified on Ni2+-charged HiTrap Chelating HP columns	Homo sapiens
histidine-tagged AP endonucleases are purified on Ni2+-charged HiTrap Chelating HP columns	Leishmania major

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	Mutation A138D has decreased AP incision activity	Leishmania major
additional information	-	Mutation D70A has decreased AP incision activity	Homo sapiens
additional information	-	The nucleotide incision repair (NIR) activity of APE1 provides an alternative and complementary repair pathway to BER	Homo sapiens
additional information	-	The specific activity is significantly increased in the case of the D70A mutant enzyme. Upon incubation at 37°C, 5 nM of purified APE1 mutant D70A remove most of the 3'-phosphoglycolate residues in 10 min, while the same enzyme amounts of APE1 converts only around 60% and 50% of the substrate into the corresponding 3'-OH form.	Homo sapiens
additional information	-	upon incubation at 37°C, 5 nM of LMAP protein remove most of the 3'-PG residues in 10 min, while the LMAP mutant A138D converts only around 50% of the substrate into the corresponding 3'-OH form	Leishmania major

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
DNA	DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase, apurinic, apyrimidinic (AP) lyases	Leishmania major	fragments of DNA	-	?
DNA	DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases	Homo sapiens	fragments of DNA	-	?
additional information	APE1 can incise DNA at the 5'-position of oxidized pyrimidine bases such as 5,6-dihydro-thymine or 5,6-dihydro-2'-deoxyuracil (DHU), thus initiating a repair process known as nucleotide incision repair (NIR)	Homo sapiens	?	-	?
additional information	APE1 has DNA 3'-phosphatase activity in vitro and 3' to 5' exonuclease activity, which could be physiologically relevant in the removal of mismatched or damaged nucleotides incorporated during the synthesis step of BER	Homo sapiens	?	-	?
additional information	In addition to the exonuclease function, human APE1 is endowed with another enzymatic activity potentially relevant for the protection against oxidative damage	Homo sapiens	?	-	?
additional information	In human cells, APE1 excises sugar fragments that block the 3'-ends thus facilitating DNA repair synthesis	Homo sapiens	?	-	?
additional information	LMAP shares with APE1 the overall 3D structure and most of the catalytic groups in their active sites and can catalyze the removal of damaged nucleotides and phosphate groups from 3'-ends more efficiently than APE1	Leishmania major	?	-	?
THF-containing oligonucleotide	AP endonuclease activity	Homo sapiens	?	-	?
THF-containing oligonucleotide	AP endonuclease activity	Leishmania major	?	-	?

Synonyms

Synonyms	Comment	Organism
APE1	-	Homo sapiens
apurinic/apyrimidinic endonuclease	-	Homo sapiens
apurinic/apyrimidinic endonuclease	-	Leishmania major
class II AP endonuclease LMAP	homolog of human Ape1	Leishmania major

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens
37	-	assay at	Leishmania major

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
additional information	-	additional information	wild-type APE1 cleaves AP sites more efficiently than D70A mutant with a kcat value for the incision of an AP site aproximately 10fold higher	Homo sapiens
1020	-	THF-containing oligonucleotide	APE1 mutant D70A, AP endonuclease activity	Homo sapiens
1140	-	DNA	LMAP mutant A138D, 3'-phosphodiesterase activity	Leishmania major
2340	-	DNA	Ape1, 3'-phosphodiesterase activity	Homo sapiens
6300	-	THF-containing oligonucleotide	LMAP mutant A138D, AP endonuclease activity	Leishmania major
10440	-	DNA	LMAP, 3'-phosphodiesterase activity	Leishmania major
12120	-	THF-containing oligonucleotide	APE1, AP endonuclease activity	Homo sapiens
16440	-	DNA	Ape mutant D70A, 3'-phosphodiesterase activity	Homo sapiens
19260	-	THF-containing oligonucleotide	LMAP, AP endonuclease activity	Leishmania major

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at (AP endonuclease activity)	Homo sapiens
8	-	assay at (endonuclease activity)	Leishmania major