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Literature summary for 4.2.99.18 extracted from

  • Kropachev, K.Y.; Zharkov, D.O.; Grollman, A.P.
    Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger (2006), Biochemistry, 45, 12039-12049.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
additional information opposite-base specificity of Nei is primarily governed by residues in the Q69-Y71 loop, both Q69-Y71 loop and the zinc finger contribute significantly to the substrate specificity of Nei Escherichia coli

Cloned(Commentary)

Cloned (Comment) Organism
into vector pET-24b, recombinant plasmids maintained in Escherichia coli XL1-Blue X cells, transfected into Escherichia L21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
deltaQLY69-71 deletion of the entire loop, interferes with eversion of the damaged base from the helix, deficient in processing damaged DNA Escherichia coli
Q261A perturbs the conserved zinc finger, deficient in processing damaged DNA, can be reductively cross-linked to damaged base-containing DNA, deficient in regenerating free enzyme from the Nei-DNA covalent complex formed during the reaction Escherichia coli
QLY69-71AAA all amino acids in the QLY loop substituted with alanines, interferes with eversion of the damaged base from the helix, deficient in processing damaged DNA Escherichia coli
R171A perturbs the conserved zinc finger, deficient in processing damaged DNA Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.000000083
-
DNA containing apurinic/apyrimidinic sites wild-type, Nei placed opposite A Escherichia coli
0.00000011
-
DNA containing apurinic/apyrimidinic sites wild-type, Nei placed opposite G Escherichia coli
0.0000023
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite G Escherichia coli
0.0000023
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite G Escherichia coli
0.0000032
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite A Escherichia coli
0.0000035
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite T Escherichia coli
0.0000044
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite C Escherichia coli
0.0000044
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite T Escherichia coli
0.0000046
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite T Escherichia coli
0.0000047
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite C Escherichia coli
0.0000066
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite C Escherichia coli
0.0000085
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite G Escherichia coli
0.000016
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite A Escherichia coli
0.000022
-
DNA containing apurinic/apyrimidinic sites mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.000028
-
DNA containing apurinic/apyrimidinic sites mutant QLY/AAA, Nei placed opposite A Escherichia coli
0.000038
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite A Escherichia coli
0.000042
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.000049
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.000054
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant deltaQLY, Nei placed opposite G Escherichia coli
0.000066
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant deltaQLY, Nei placed opposite A Escherichia coli
0.000077
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.00014
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite C Escherichia coli
0.00016
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite T Escherichia coli
0.00016
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite A Escherichia coli
0.00019
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant QLY/AAA, Nei placed opposite T Escherichia coli
0.00021
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.00024
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.00026
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite A Escherichia coli
0.00027
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite C Escherichia coli
0.00058
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite G Escherichia coli
0.00061
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite A Escherichia coli
0.00068
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite C Escherichia coli
0.0013
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite T Escherichia coli
0.0014
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite T Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+
-
Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P50465
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3'
-
Escherichia coli ?
-
?
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3'
-
Escherichia coli ?
-
?
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3'
-
Escherichia coli ?
-
?
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3'
-
Escherichia coli ?
-
?
DNA containing apurinic/apyrimidinic sites
-
Escherichia coli ?
-
?

Synonyms

Synonyms Comment Organism
endonuclease VIII
-
Escherichia coli
Nei
-
Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0000048
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant deltaQLY, Nei placed opposite G Escherichia coli
0.000013
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite C Escherichia coli
0.000015
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.000023
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite A Escherichia coli
0.000065
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.000067
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant Q261A, Nei placed opposite T Escherichia coli
0.00007167
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite A Escherichia coli
0.000583
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite A Escherichia coli
0.0015
-
DNA containing apurinic/apyrimidinic sites mutant QLY/AAA, Nei placed opposite A Escherichia coli
0.00183
-
DNA containing apurinic/apyrimidinic sites mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.0025
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant deltaQLY, Nei placed opposite A Escherichia coli
0.003167
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite T Escherichia coli
0.00567
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.00583
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant Q261A, Nei placed opposite G Escherichia coli
0.00783
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite A Escherichia coli
0.008167
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite C Escherichia coli
0.00883
-
DNA containing apurinic/apyrimidinic sites wild-type, Nei placed opposite G Escherichia coli
0.01067
-
DNA containing apurinic/apyrimidinic sites wild-type, Nei placed opposite A Escherichia coli
0.0112
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite T Escherichia coli
0.0115
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite C Escherichia coli
0.01167
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite T Escherichia coli
0.013
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' mutant QLY/AAA, Nei placed opposite C Escherichia coli
0.01317
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite G Escherichia coli
0.01583
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite C Escherichia coli
0.016
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant QLY/AAA, Nei placed opposite G Escherichia coli
0.02167
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' mutant QLY/AAA, Nei placed opposite T Escherichia coli
0.023
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite A Escherichia coli
0.023
-
5'-CTCTCCCTTC-8-oxo-7,8-dihydroguanine-CTCCTTTCCTCT-3' wild-type, Nei placed opposite T Escherichia coli
0.03
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite G Escherichia coli
0.035
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite A Escherichia coli
0.04167
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite C Escherichia coli
0.0483
-
5'-GACAAGCGCAG-(5R,6S)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite T Escherichia coli
0.0567
-
5'-GACAAGCGCAG-(5S,6R)-2'-deoxy-5,6-dihydroxyuridine-CAGCCGAACAC-3' wild-type, Nei placed opposite G Escherichia coli
0.07167
-
5'-CTCTCCCTTC-5,6-dihydrouracil-CTCCTTTCCTCT-3' wild-type, Nei placed opposite G Escherichia coli