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Literature summary for 3.1.26.4 extracted from

  • Haruki, M.; Nogawa, T.; Hirano, N.; Chon, H.; Tsunaka, Y.; Morikawa, M.; Kanaya, S.
    Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H (2000), Protein Eng., 13, 881-886.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzymes in Escherichia coli strain HB101 Thermus thermophilus
overexpression of wild-type and mutant enzymes in strain HB101 Escherichia coli

Protein Variants

Protein Variants Comment Organism
C13A/C63A/C133A/E135C site-directed mutagenesis, 37% activity compared to the wild-type enzyme Escherichia coli
C13S/C63A/R135C site-directed mutagenesis, 6.9% activity compared to the wild-type enzyme Thermus thermophilus
C63A site-directed mutagenesis, 94% activity compared to the wild-type enzyme Thermus thermophilus
C63A/R135C site-directed mutagenesis, 44% activity compared to the wild-type enzyme Thermus thermophilus
additional information construction of DNA-linked mutant enzyme, by cross-linking of the 15mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA, i.e. PPT-RNA, to the thiol group of Cys13 or Cys135 of the enzyme, mutants shows 0.69% activity compared to the wild-type enzyme Thermus thermophilus
additional information construction of DNA-linked mutant enzyme, by cross-linking of the 15mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA, i.e. PPT-RNA, to the thiol group of Cys135 of the enzyme, mutant shows 4.2% activity compared to the wild-type enzyme Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-
Thermus thermophilus
-
-
-

Reaction

Reaction Comment Organism Reaction ID
Endonucleolytic cleavage to a 5'-phosphomonoester active site residues are Asp10, Gu48, Asp70, His124, Asp134 Thermus thermophilus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
activity of wild-type and mutant enzymes, determination by spectrophotometric assay, at different temperatures Thermus thermophilus
additional information
-
activity of wild-type and mutant enzymes, determination by spectrophotometric assay, at different temperatures Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
PPT-RNA + H2O single-stranded, the DNA-linked enzyme mutant shows highly reduced activity compared to the wild-type enzyme, specific cleavage of the 15mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA Thermus thermophilus ?
-
?
PPT-RNA + H2O single-stranded, the DNA-linked enzyme mutant shows highly reduced activity compared to the wild-type enzyme, specific cleavage of the 15mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA Escherichia coli ?
-
?
RNA*DNA hybrid + H2O specific for Thermus thermophilus ?
-
?
RNA*DNA hybrid + H2O specific for Escherichia coli ?
-
?

Subunits

Subunits Comment Organism
More three-dimensional structure of wild-type enzyme Thermus thermophilus

Synonyms

Synonyms Comment Organism
endoribonuclease H (calf thymus)
-
Thermus thermophilus
ERNH
-
Escherichia coli
hybrid nuclease
-
Thermus thermophilus
hybrid ribonuclease
-
Thermus thermophilus
hybridase
-
Thermus thermophilus
hybridase (ribonuclease H)
-
Thermus thermophilus
ribonuclease H
-
Thermus thermophilus
ribonuclease H
-
Escherichia coli
RNA*DNA hybrid ribonucleotiohydrolase
-
Thermus thermophilus
RNase H
-
Thermus thermophilus
RNase H
-
Escherichia coli
RNase HI
-
Thermus thermophilus
RNase HI
-
Escherichia coli
TRNH
-
Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at, substrate RNA*DNA hybrid Thermus thermophilus
30
-
assay at, substrate RNA*DNA hybrid Escherichia coli
50
-
DNA-C135-linked mutant, substrate PPT-RNA Escherichia coli
65
-
assay at, substrate PPT-RNA Escherichia coli
65
-
wild-type enzyme, assay at, substrate PPT-RNA, DNA-C135-linked mutant enzyme shows optimal activity with this substrate at 65°C Thermus thermophilus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
DNA-linked mutant, loss of 50% activity after 15 min Escherichia coli
60
-
DNA-linked mutant, complete inactivation after 15 min Escherichia coli
90
-
DNA-linked mutant is almost fully stable after 15 min Thermus thermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli
9
-
assay at Thermus thermophilus