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Literature summary for 3.1.26.3 extracted from
- Biochemical properties of Bacillus Calmette Guerin ribonuclease III (2016), J. Basic Microbiol., 56, 392-404 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene rnc, recombinant expression of wild-type and mutant GST-tagged enzymes in Escherichia coli strain Bl21(DE3) | Mycobacterium tuberculosis variant bovis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D48A | site-directed mutagenesis, catalytically inactive mutant | Mycobacterium tuberculosis variant bovis |
| E44A | site-directed mutagenesis, catalytically inactive mutant | Mycobacterium tuberculosis variant bovis |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| EDTA | complete inhibition | Mycobacterium tuberculosis variant bovis |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics | Mycobacterium tuberculosis variant bovis |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Co2+ | required, activates | Mycobacterium tuberculosis variant bovis | |
| Mg2+ | required, activates | Mycobacterium tuberculosis variant bovis | |
| Mn2+ | required, activates | Mycobacterium tuberculosis variant bovis | |
| additional information | the enzyme recognizes RNA motifs and cleaves substrates at specific sites in a divalent-metal-ion-dependent manner. The RNA cleavage activity of the enzyme can be supported by Mg2+, Mn2+, and Co2+ and enhanced with the increasing salt concentration. BCGRNase III can exploit Mg2+, Mn2+, and Co2+, but not Ni2+, Ca2+, Zn2+, and Cu2+, as a cofactors to show RNA cleavage activity. The maximum activity of the enzyme supported by Mn2+ ions is 0.12fold higher than that supported by Mg2+ ions | Mycobacterium tuberculosis variant bovis |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Mycobacterium tuberculosis variant bovis | A0A0H3M8A2 | live attenuated Bacillus Calmette Guerin strain, Pasteur strain | - |
| Mycobacterium tuberculosis variant bovis Pasteur 1173P2 | A0A0H3M8A2 | live attenuated Bacillus Calmette Guerin strain, Pasteur strain | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant wild-type and mutant GST-tagged enzymes from Escherichia coli strain Bl21(DE3) by glutathione affinity chromatography, tag cleavage by thrombin, and 4-aminobenzamidine affinity chromatography to remove thrombin, followed by gel filtration, to homogeneity | Mycobacterium tuberculosis variant bovis |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| Endonucleolytic cleavage to a 5'-phosphomonoester | catalytic mechanism of RNase III, overview. RNase III activates water as a nucleophile to hydrolyze target site phosphodiesters, creating 3'-hydroxyl, 5'-phosphomonoester product termini, Mg2+ serves as the ion facilitating departure of the 3'-oxygen atom, while Mg2+ binds and activates the water nucleophile. And both metal ions may coordinate to both the nucleophile and the nonbridging phosphoryl oxygen to stabilize the in-line geometry required for phosphoryl transfer reactions | Mycobacterium tuberculosis variant bovis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | the recombinant RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites, remnant endogenous ribonucleases from the expression host have no effect on cleavage assays. BCG-16S [hp] RNA is synthesized using 2.5 U/ml T7 RNA polymerase at 42°C for 4 h. The specific activity of the alpha-32P-UTP in the transcription reactions is 200 Ci/mol | Mycobacterium tuberculosis variant bovis | ? | - |
? | |
| additional information | the recombinant RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites, remnant endogenous ribonucleases from the expression host have no effect on cleavage assays. BCG-16S [hp] RNA is synthesized using 2.5 U/ml T7 RNA polymerase at 42°C for 4 h. The specific activity of the alpha-32P-UTP in the transcription reactions is 200 Ci/mol | Mycobacterium tuberculosis variant bovis Pasteur 1173P2 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 52000, recombinant GST-tagged RNase III, SDS-PAGE, x * 26000, recombinant detagged enzyme, SDS-PAGE | Mycobacterium tuberculosis variant bovis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| BCG-RNase III | - |
Mycobacterium tuberculosis variant bovis |
| RNase III | - |
Mycobacterium tuberculosis variant bovis |
| rnc | - |
Mycobacterium tuberculosis variant bovis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
- |
Mycobacterium tuberculosis variant bovis |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | 55 | activity range, recombinant enzyme, inactivation at 60°C and above | Mycobacterium tuberculosis variant bovis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
- |
Mycobacterium tuberculosis variant bovis |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 10 | activity range, recombinant enzyme | Mycobacterium tuberculosis variant bovis |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | the enzyme is a member of the ribonuclease III (RNase III) family | Mycobacterium tuberculosis variant bovis |
| malfunction | in enzyme mutants E44A and D48A, the bond between the residue E44 and Mg2+ is broken and the bond between the residue D48 and Mg2+ interrupted. The proper positioning of Mg2+ ion in catalytic centre fails because of these broken bonds. The enzyme is deactivated by preventing the formation of trigonal bipyramidal pentavalent phosphorane intermediate | Mycobacterium tuberculosis variant bovis |
| additional information | members of ribonuclease III (RNase III) family recognize RNA motifs and cleave substrates at specific sites in a divalent-metal-ion-dependent manner. The residues E44 and D48 in BCG-RNase III are highly conserved and essential for catalytic activity | Mycobacterium tuberculosis variant bovis |
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