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Literature summary extracted from

  • Georgiadis, M.M.; Luo, M.; Gaur, R.K.; Delaplane, S.; Li, X.; Kelley, M.R.
    Evolution of the redox function in mammalian apurinic/apyrimidinic endonuclease (2008), Mutat. Res., 643, 54-63.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.99.18 expression vectors encoding the full-length zApe and zApe (33-310) proteins in pET15b are constructed using standard subcloning techniques and verified by DNA sequencing. The zApe vectors are transformed into Rosetta (DE3) Escherichia coli, cultures are grown in LB media Danio rerio
4.2.99.18 The wild-type and mutant hApe1 proteins are expressed as GST-fusions in Escherichia coli Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.99.18 crystals of zApe (74 mg/ml in 10 mM HEPES, pH 7.5 buffer) are grown by hanging drop vapor diffusion at 20°C using 200 mM ammonium acetate, 100 mM Bis-Tris pH 5.5, 7% PEG 3350, 2% glycerol and 0.8 mM lead acetate as the precipitating solution. The structure is solved at a resolution of 49.15-2.3 A Danio rerio
4.2.99.18 mutant C65A hApe1 protein is diluted to 10 mg/ml in 10 mM HEPES pH 7.5 and then crystallized by hanging drop vapor diffusion using 10 mM MES pH 6.0, 7.5 mM Sm(OAc)3, 4% dioxane, 10-20% PEG 8000 as the precipitating solution at 20°C. The structure is solved at a resolution of 33.0-1.9 A Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
4.2.99.18 C138A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 C208A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 C296A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 C310A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 C65A site-directed mutagenesis of a pET28A vector encoding human Ape1 (40-318) is performed, mutant is redox inactive Homo sapiens
4.2.99.18 C93A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 C99A stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active Homo sapiens
4.2.99.18 T58C Mutant enzyme is redox active. Site-directed mutagenesis is performed on the full-length zebrafish Ape pET15b vector using Stratagene Quikchange kit and verified by DNA sequencing. The T58C zApe vector is then transformed into Rosetta Escherichia coli (DE3). Stable ovarian cancer Skov-3X cells with the NFkappaB-Luc gene are cotransfected with plasmid pcDNA-mutant and a Renilla luciferase control reporter vector pRL-CMV using lipofectamine. Danio rerio

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.99.18 DNA Homo sapiens
-
 fragments of DNA
-
 ?
4.2.99.18 DNA Danio rerio
-
 fragments of DNA
-
 ?
4.2.99.18 additional information Homo sapiens enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others ?
-
 ?
4.2.99.18 additional information Danio rerio enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.99.18 Danio rerio
-
 zebrafish
-
4.2.99.18 Homo sapiens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.99.18 First step is a Ni-NTA affinity chromatography. Then the Ni-NTA fractions containing zApe are combined and loaded on a pre-equilibrated MonoQ column after dilution to a final NaCl concentration of 50 mM. The protein is eluted using a linear NaCl gradient, and fractions containing the zApe are combined, digested with thrombin, and then subjected again to a final MonoQ column. The purified proteins are concentrated to 50-75 mg/ml. The mutant T58C is purified by using an Q-sepharose column as ion exchanger. Danio rerio
4.2.99.18 Purification is done using the standard glutathione affinity purification protocol. The C65A mutant is purified as follows: cell suspension is centrifuged, supernatant is loaded on a Ni-NTA column and the protein is eluted with a linear imidazole gradient. Fractions containing C65A hApe1 are further purified on an S-Sepharose column with a linear NaCl gradient. Homo sapiens

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.99.18 additional information
-
 Cell-based analysis of redox activity for hApe1 Cys-mutants using an in vitro EMSA redox assay in which reduction of the trancription factor AP-1 by hApe1 results in a shifted band. Only C65A hApe1 is found to be redox inactive. Homo sapiens
4.2.99.18 additional information
-
 T58C zApe has near wild-type human Ape1 redox activity in the transactivation assay Danio rerio

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.99.18 DNA
-
 Homo sapiens fragments of DNA
-
 ?
4.2.99.18 DNA
-
 Danio rerio fragments of DNA
-
 ?
4.2.99.18 additional information enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others Homo sapiens ?
-
 ?
4.2.99.18 additional information enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others Danio rerio ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.99.18 monomer C65A hApe1 in solution, determined by size exclusion chromatography Homo sapiens
4.2.99.18 monomer zApe in solution, determined by gel filtration chromatography Danio rerio

Synonyms

EC Number Synonyms Comment Organism
4.2.99.18 Ape
-
 Homo sapiens
4.2.99.18 Ape
-
 Danio rerio
4.2.99.18 apurinic/apyrimidinic endonuclease
-
 Homo sapiens
4.2.99.18 apurinic/apyrimidinic endonuclease
-
 Danio rerio
4.2.99.18 hAPE
-
 Homo sapiens
4.2.99.18 hAPE1
-
 Homo sapiens
4.2.99.18 zApe
-
 Danio rerio