Literature summary for 3.1.26.4 extracted from

Chapados, B.R.; Chai, Q.; Hosfield, D.J.; Qiu, J.; Shen, B.; Tainer, J.A.
Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication (2001), J. Mol. Biol., 307, 541-556.

Cloned(Commentary)

Cloned (Comment)	Organism
type 2 enzyme, overexpression of wild-type in Escherichia coli BL21(DE3), and of selenomethionine mutant in Escherichia coli strain B834, a methionine-auxotroph BL21(DE3) derivative	Archaeoglobus fulgidus

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystallization of 40 mg/ml purified recombinant wild-type type 2 enzyme, and of recombinant selenomethionine enzyme mutant, with or without bound metal ions, hanging drop vapour diffusion method, mixing with equal volume of precipitant solution containing 10% PEG monomethylether 5000, 10% butanol, 20 mM sodium citrate, 100 mM Tris, pH 8.5, 3 days, formation of a cobalt-hexammine chloride complex, X-ray diffraction structure determination and analysis at 1.95-2.7 A resolution	Archaeoglobus fulgidus

Crystallization (Comment)

Organism

crystallization of 40 mg/ml purified recombinant wild-type type 2 enzyme, and of recombinant selenomethionine enzyme mutant, with or without bound metal ions, hanging drop vapour diffusion method, mixing with equal volume of precipitant solution containing 10% PEG monomethylether 5000, 10% butanol, 20 mM sodium citrate, 100 mM Tris, pH 8.5, 3 days, formation of a cobalt-hexammine chloride complex, X-ray diffraction structure determination and analysis at 1.95-2.7 A resolution

Archaeoglobus fulgidus

Protein Variants

Protein Variants	Comment	Organism
K143A	site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme	Archaeoglobus fulgidus
R146A	site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme	Archaeoglobus fulgidus
R46A	site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme	Archaeoglobus fulgidus
Y164A	site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme	Archaeoglobus fulgidus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00006	-	RNA-DNA*DNA hybrid	recombinant wild-type enzyme, pH 8.0, 30°C	Archaeoglobus fulgidus
0.0016	-	RNA-DNA*DNA hybrid	recombinant mutant R146A, pH 8.0, 30°C	Archaeoglobus fulgidus
0.00188	-	RNA-DNA*DNA hybrid	recombinant mutant K143A, pH 8.0, 30°C	Archaeoglobus fulgidus
0.00268	-	RNA-DNA*DNA hybrid	recombinant mutant Y164A, pH 8.0, 30°C	Archaeoglobus fulgidus
0.0036	-	RNA-DNA*DNA hybrid	recombinant mutant R46A, pH 8.0, 30°C	Archaeoglobus fulgidus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	dependent on	Archaeoglobus fulgidus
additional information	metal coordination in the active site	Archaeoglobus fulgidus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
RNA-DNA*DNA hybrid + H2O	Archaeoglobus fulgidus	RNA primer recognition and removal during DNA replication	DNA*DNA + 5'-phosphomononucleotides	-	?

Organism

Organism	UniProt	Comment	Textmining
Archaeoglobus fulgidus	O29634	type 2 enzyme	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzymes from Escherichia coli	Archaeoglobus fulgidus

Reaction

Reaction	Comment	Organism	Reaction ID
Endonucleolytic cleavage to a 5'-phosphomonoester	active site structure, substrate binding groove, molecular mechanism for specific RNA*DNA hybrid substrate recognition, binding, and cleavage, a general nuclease activity is necessary for catalysis	Archaeoglobus fulgidus	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	cleavage specificity	Archaeoglobus fulgidus	?	-	?
RNA-DNA single-stranded chimera + H2O	-	Archaeoglobus fulgidus	?	-	?
RNA-DNA*DNA hybrid + H2O	RNA primer recognition and removal during DNA replication	Archaeoglobus fulgidus	DNA*DNA + 5'-phosphomononucleotides	-	?
RNA-DNA*DNA hybrid + H2O	7 M urea-denatured 18mer, no activity with untreated hybrid, specific removal of the RNA portion of the duplex	Archaeoglobus fulgidus	8mer oligonucleotide	-	?
RNA-DNA*DNA hybrid + H2O	7 M urea-denatured 37mer, no activity with untreated hybrid, specific removal of the RNA portion of the duplex	Archaeoglobus fulgidus	oligonucleotides	a 20mer, a 10mer, a 9mer, and a 7mer	?

Subunits

Subunits	Comment	Organism
More	two-domain architecture	Archaeoglobus fulgidus

Synonyms

Synonyms	Comment	Organism
More	the enzyme belongs to the RNase H nuclease family	Archaeoglobus fulgidus
ribonuclease H	-	Archaeoglobus fulgidus
RNase H	-	Archaeoglobus fulgidus
RNase HII	-	Archaeoglobus fulgidus
type 2 RNase H	-	Archaeoglobus fulgidus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Archaeoglobus fulgidus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.133	-	RNA-DNA*DNA hybrid	recombinant wild-type enzyme, pH 8.0, 30°C	Archaeoglobus fulgidus
0.192	-	RNA-DNA*DNA hybrid	recombinant mutant R146A, pH 8.0, 30°C	Archaeoglobus fulgidus
0.2	-	RNA-DNA*DNA hybrid	recombinant mutant R46A and K143A, pH 8.0, 30°C	Archaeoglobus fulgidus
0.208	-	RNA-DNA*DNA hybrid	recombinant mutant Y164A, pH 8.0, 30°C	Archaeoglobus fulgidus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Archaeoglobus fulgidus