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Literature summary for 4.6.1.16 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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
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

Protein Variants Comment Organism
H133A site-directed mutagenesis, crystal structure determination Aeropyrum pernix
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
K44A site-directed mutagenesis, the mutant shows a severe loss of enzyme activity, crystal structure determination Aeropyrum pernix
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
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

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 ?
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?
additional information Archaeoglobus fulgidus AFU-EndA has narrow substrate specificity, overview ?
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?

Organism

Organism UniProt Comment Textmining
Aeropyrum pernix Q9YE85
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Archaeoglobus fulgidus
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Purification (Commentary)

Purification (Comment) Organism
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
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 ?
-
?
additional information AFU-EndA has narrow substrate specificity, overview Archaeoglobus fulgidus ?
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?

Subunits

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

Synonyms

Synonyms Comment Organism
EndA
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Archaeoglobus fulgidus
EndA
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Aeropyrum pernix
splicing endonuclease
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Archaeoglobus fulgidus
splicing endonuclease
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Aeropyrum pernix

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
65
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assay at Archaeoglobus fulgidus
65
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assay at Aeropyrum pernix

pH Optimum

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

General Information

General Information Comment Organism
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
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