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Literature summary extracted from

  • Hirata, A.; Kitajima, T.; Hori, H.
    Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop (2011), Nucleic Acids Res., 39, 9376-9389.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.6.1.16 overexpression of EndA in Escherichia coli strain Rosetta 2(DE3) Archaeoglobus fulgidus
4.6.1.16 overexpression of wild-type and mutant EndAs in Escherichia coli strain Rosetta 2(DE3) Aeropyrum pernix

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.6.1.16 purified recombinant AFU-EndA, hanging-drop vapor diffusion method, 10 mg/ml protein solution is mixed in equal volumes with reservoir solution containing 2.2 M ammonium sulfate, 0.2 M potassium sodium tartrate tetrahydrate and 0.1 M sodium citrate, pH 5.6, several days, equilibration over 0.5 ml reservoir solution, 22°C, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacement Archaeoglobus fulgidus
4.6.1.16 purified recombinant wild-type and mutant APE-EndAs, hanging-drop vapor diffusion method, 10 mg/ml protein solution is mixed in equal volumes with reservoir solution containing 0.25 M ammonium sulfate, 0.1 M sodium citrate, pH 5.6, 0.9 M lithium sulfate and 1 mM MgCl2, 1 day, equilibration over 0.5 ml reservoir solution, 22°C, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacement Aeropyrum pernix

Protein Variants

EC Number Protein Variants Comment Organism
4.6.1.16 H133A site-directed mutagenesis, crystal structure determination Aeropyrum pernix
4.6.1.16 K179A site-directed mutagenesis, the mutant removes the BHB intron from the anticodon loop and the D-loop. However, AFU-CSL K179A can not remove the BHL intron correctly Archaeoglobus fulgidus
4.6.1.16 K44A site-directed mutagenesis, the mutant shows a severe loss of enzyme activity, crystal structure determination Aeropyrum pernix
4.6.1.16 additional information introduction of the Crenarchaea specific loop, CSL, into AFU-EndA enhances its intron-cleaving activity irrespective of the position or motif of the intron Archaeoglobus fulgidus
4.6.1.16 R46A site-directed mutagenesis, the substitution of R46 residue with alanine does not affect its substrate selectivity significantly, crystal structure determination Aeropyrum pernix

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.6.1.16 Mg2+ required Archaeoglobus fulgidus
4.6.1.16 Mg2+ required Aeropyrum pernix

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.6.1.16 additional information Aeropyrum pernix Aeropyrum pernix expresses precursor-tRNAs with canonical or non-canonical introns at various positions. APE splicing endonuclease removes both types of introns, including the non-canonical introns, without any nucleotide modification. E.g. three tRNAThr species are transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them are cleaved from standard and non-standard positions, APE-EndA has broad substrate specificity, overview ?
-
 ?
4.6.1.16 additional information Archaeoglobus fulgidus AFU-EndA has narrow substrate specificity, overview ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.6.1.16 Aeropyrum pernix Q9YE85
-
-
4.6.1.16 Archaeoglobus fulgidus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.6.1.16 recombinant EndA from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70°C for 30 min, followed by heparin affinity chromatography and gel filtration Archaeoglobus fulgidus
4.6.1.16 recombinant wild-type and mutant EndAs from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70°C for 30 min, followed by heparin affinity chromatography and gel filtration Aeropyrum pernix

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.6.1.16 additional information Aeropyrum pernix expresses precursor-tRNAs with canonical or non-canonical introns at various positions. APE splicing endonuclease removes both types of introns, including the non-canonical introns, without any nucleotide modification. E.g. three tRNAThr species are transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them are cleaved from standard and non-standard positions, APE-EndA has broad substrate specificity, overview Aeropyrum pernix ?
-
 ?
4.6.1.16 additional information AFU-EndA has narrow substrate specificity, overview Archaeoglobus fulgidus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.6.1.16 More structure comparisons of EndA from Aeropyrum pernix and from Archaeaoglobus fulgidus, overview Archaeoglobus fulgidus
4.6.1.16 More structure comparisons of EndA from Aeropyrum pernix and from Archaeaoglobus fulgidus, overview Aeropyrum pernix

Synonyms

EC Number Synonyms Comment Organism
4.6.1.16 EndA
-
 Archaeoglobus fulgidus
4.6.1.16 EndA
-
 Aeropyrum pernix
4.6.1.16 splicing endonuclease
-
 Archaeoglobus fulgidus
4.6.1.16 splicing endonuclease
-
 Aeropyrum pernix

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.6.1.16 65
-
 assay at Archaeoglobus fulgidus
4.6.1.16 65
-
 assay at Aeropyrum pernix

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.6.1.16 7.5
-
 assay at Archaeoglobus fulgidus
4.6.1.16 7.5
-
 assay at Aeropyrum pernix

General Information

EC Number General Information Comment Organism
4.6.1.16 additional information APE-EndA possesses a Crenarchaea specific loop, which is responsible for the broad substrate specificity of APE-EndA. Lys44 in CSL functions as the RNA recognition site Aeropyrum pernix
4.6.1.16 additional information introduction of the Crenarchaea specific loop, CSL, eg.e from Aeropyrum pernix EndA, into AFU-EndA enhances its intron-cleaving activity irrespective of the position or motif of the intron Archaeoglobus fulgidus