Literature summary for 3.1.26.4 extracted from

Chon, H.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus: a new type 2 RNase H with TBP-like substrate-binding domain at the N terminus (2006), J. Mol. Biol., 356, 165-178.

Cloned(Commentary)

Cloned (Comment)	Organism
complementation assay using the enzyme-deficient Escherichia coli strain MIC2067, which shows an RNase H-dependent temperature-sensitive growth phenotype, by wild-type and mutant enzymes, no complementation by the C domian mutant, overview	Geobacillus stearothermophilus
gene rnhC, overexpression of the soluble enzyme in Escherichia coli strain MIC2067(DE3)	Geobacillus stearothermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme, free or bound to Mg2+ or Mn2+, 6.4-8.0 mg/ml protein in 85 mM Tris-HCl, pH 8.5, 0.17 M lithium sulfate, 25.5% w/v PEG 4000, and 15% v/v glycerol, metal binding by soaking of crystals in 1 mM Mg2+ or Mn2+ containing solution for 24 h, heavy atom derivatization with 10 mM CH3HgCl or K2PtCl6, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution, modeling	Geobacillus stearothermophilus
RNase HIII in both metal-free and metal-bound form, 6.4-8.0 mg/ml protein, native enzyme crystals from 85 mM TrisHCl, pH 8.5, 0.17 M lithium sulfate, 25.5% w/v PEG 4000, and 15% v/v glycerol, soaking of crystals in a solution containing 50 mM Mg2+ or Mn2+, preparation of heavy-metal derivatives, X-ray diffraction structure determiation and analysis at 2.1-2.6 A resolution, modelling	Geobacillus stearothermophilus

Crystallization (Comment)

Organism

purified recombinant enzyme, free or bound to Mg2+ or Mn2+, 6.4-8.0 mg/ml protein in 85 mM Tris-HCl, pH 8.5, 0.17 M lithium sulfate, 25.5% w/v PEG 4000, and 15% v/v glycerol, metal binding by soaking of crystals in 1 mM Mg2+ or Mn2+ containing solution for 24 h, heavy atom derivatization with 10 mM CH3HgCl or K2PtCl6, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution, modeling

Geobacillus stearothermophilus

RNase HIII in both metal-free and metal-bound form, 6.4-8.0 mg/ml protein, native enzyme crystals from 85 mM TrisHCl, pH 8.5, 0.17 M lithium sulfate, 25.5% w/v PEG 4000, and 15% v/v glycerol, soaking of crystals in a solution containing 50 mM Mg2+ or Mn2+, preparation of heavy-metal derivatives, X-ray diffraction structure determiation and analysis at 2.1-2.6 A resolution, modelling

Geobacillus stearothermophilus

Protein Variants

Protein Variants	Comment	Organism
D202A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
D202A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
D97A	site-directed mutagenesis, inactive mutant	Geobacillus stearothermophilus
D97A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
E232A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
E98A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
E98A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Geobacillus stearothermophilus
additional information	construction of deletion mutants of RNase HIII by truncation of the N-domain, residues 1-79, the C-domain, residues 80-310, the RNase HIIIDELTAC domain, residues 1-299, and C-domainDELTAC, residues 80-299, the mutant show reduced activity, overview	Geobacillus stearothermophilus

Metals/Ions

Metals/Ions	Comment	Organism
Mg2+	required, best at 50 mM	Geobacillus stearothermophilus
Mg2+	the enzyme requires Mn2+ or Mg2+ ions, Mg2+ is preferred, coordinated with Asp97, Glu98, and Asp202	Geobacillus stearothermophilus
Mn2+	less active than Mg2+, best at 10 mM	Geobacillus stearothermophilus
Mn2+	the enzyme requires Mn2+ or Mg2+ ions, Mg2+ is preferred, coordinated with Asp97, Glu98, and Asp202	Geobacillus stearothermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
DNA-RNA hybrid + H2O	Geobacillus stearothermophilus	-	DNA + 5'-phosphonucleotides	-	?

Organism

Organism	UniProt	Comment	Textmining
Geobacillus stearothermophilus	Q6L6Q4	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
Endonucleolytic cleavage to a 5'-phosphomonoester	active site and substrate binding site structures	Geobacillus stearothermophilus	a
Endonucleolytic cleavage to a 5'-phosphomonoester	four acidic active-site residues of Bst-RNase HIII: Asp97, Glu98, Asp202, and Glu232, substrate binding and site structure, active site structure and reaction mechanism, overview	Geobacillus stearothermophilus	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.001	-	below, mutant D97A, with 50 mM Mg2+ or 10 mM Mn2+	Geobacillus stearothermophilus
0.0046	-	mutant E98A, with 10 mM Mn2+	Geobacillus stearothermophilus
0.0051	-	mutant E98A, with 10 mM Mg2+	Geobacillus stearothermophilus
0.03	-	mutant D202A, with 10 mM Mn2+	Geobacillus stearothermophilus
0.046	-	mutant D202A, with 10 mM Mg2+	Geobacillus stearothermophilus
0.3	-	mutant E232A, with 10 mM Mn2+	Geobacillus stearothermophilus
0.42	-	mutant E232A, with 10 mM Mg2+	Geobacillus stearothermophilus
1.1	-	wild-type enzyme, with 10 mM Mn2+	Geobacillus stearothermophilus
1.9	-	wild-type enzyme, with 50 mM Mg2+	Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
12 basepair DNA-DNA duplex + H2O	oligomeric substrate, cleavage at multiple sites, product identification	Geobacillus stearothermophilus	?	-	?
29 basepair DNA-RNA-DNA/DNA + H2O	oligomeric substrate, cleavage mainly in the middle of the tetraribonucleotide, product identification	Geobacillus stearothermophilus	?	-	?
DNA-RNA hybrid + H2O	-	Geobacillus stearothermophilus	DNA + 5'-phosphonucleotides	-	?
DNA-RNA hybrid + H2O	3H-labeled M13 DNA/RNA hybrid substrate, the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding to a higher extent than the latter, overview	Geobacillus stearothermophilus	DNA + 5'-phosphonucleotides	-	?
M13 DNA-RNA hybrid + H2O	-	Geobacillus stearothermophilus	?	-	?

Subunits

Subunits	Comment	Organism
More	the enzyme contains a TBP-like substrate-binding domain at the N terminus	Geobacillus stearothermophilus
More	RNase HIII consists of the N-terminal domain and C-terminal RNase H domain, the enzyme contains a TBP-like substrate-binding domain at the N terminus	Geobacillus stearothermophilus

Synonyms

Synonyms	Comment	Organism
RNase HIII	-	Geobacillus stearothermophilus
type 2 RNase H	-	Geobacillus stearothermophilus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Geobacillus stearothermophilus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Geobacillus stearothermophilus
8.5	9	-	Geobacillus stearothermophilus