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Literature summary for 4.6.1.16 extracted from

  • Okuda, M.; Shiba, T.; Inaoka, D.K.; Kita, K.; Kurisu, G.; Mineki, S.; Harada, S.; Watanabe, Y.; Yoshinari, S.
    A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity (2011), J. Mol. Biol., 405, 92-104.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of His6-tagged wild-type and mutant EndAs in Escherichi coli strain Rosetta 2(DE3) Aeropyrum pernix

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type and mutant EndAs, method screening, sitting drop vapor diffusion technique, mixing of 0.001 ml protein solution with 0.001 ml reservoir solution containing 0.2 M NaCl, 0.1 M phosphate-citrate, pH 4.2, and 10% w/v PEG 3000, equilibration over 0.1 ml reservoir solution, 22┬░C, X-ray diffraction structure determmination and analysis at 1.7-2.3 A resolution, molecular replacement Aeropyrum pernix

Protein Variants

Protein Variants Comment Organism
D49A site-directed mutagenesis, the mutation does not affect the enzyme activity Aeropyrum pernix
E43A site-directed mutagenesis, the mutation does not affect the enzyme activity Aeropyrum pernix
E51A site-directed mutagenesis, the mutation does not affect the enzyme activity Aeropyrum pernix
F50A site-directed mutagenesis, the mutant shows reduced enzyme activity compared to the wild-type enzyme Aeropyrum pernix
H133A site-directed mutagenesis, a catalytic site mutant, crystal structure determination Aeropyrum pernix
K44A site-directed mutagenesis, the mutant shows almost no enzyme activity Aeropyrum pernix
P45A site-directed mutagenesis, the mutant shows reduced enzyme activity compared to the wild-type enzyme Aeropyrum pernix
R46A site-directed mutagenesis, the mutant shows reduced enzyme activity compared to the wild-type enzyme Aeropyrum pernix

Organism

Organism UniProt Comment Textmining
Aeropyrum pernix Q9YBF1
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Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant EndAs from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70┬░C for 30 min, followed by metal affinity chromatography and gel filtration Aeropyrum pernix

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information Sulfolobus tokodaii tRNATrp precursor as a substrate Aeropyrum pernix ?
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?

Synonyms

Synonyms Comment Organism
EndA
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Aeropyrum pernix
splicing endonuclease
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Aeropyrum pernix

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
70
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assay at Aeropyrum pernix

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
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assay at Aeropyrum pernix

General Information

General Information Comment Organism
evolution the crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit Aeropyrum pernix
additional information EndA from Aeropyrum pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs, the conserved lysine residue Lys44 in the loop is important for endonuclease activity, substrate docking modeling, overview Aeropyrum pernix