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.

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

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	?	-	?
additional information	Archaeoglobus fulgidus	AFU-EndA has narrow substrate specificity, overview	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Aeropyrum pernix	Q9YE85	-	-
Archaeoglobus fulgidus	-	-	-

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	?	-	?

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	-	Archaeoglobus fulgidus
EndA	-	Aeropyrum pernix
splicing endonuclease	-	Archaeoglobus fulgidus
splicing endonuclease	-	Aeropyrum pernix

Temperature Optimum [°C]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	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