Cloned (Comment) | Organism |
---|---|
genes rnpA and rnpB, recombinant expression of GST-tagged wild-type and mutant proteins and RNA in Escherichia coli strain BL21(DE3)pLysS | Thermotoga maritima |
Protein Variants | Comment | Organism |
---|---|---|
F17A | site-directed mutagenesis of the protein part, the mutant shows 95% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
F17A/U52C | site-directed mutagenesis of the protein part (F17A) and the RNA part (U52C), the mutant shows over 99% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
F21A | site-directed mutagenesis of the protein part, the mutant shows 80% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
K51A | site-directed mutagenesis of the protein part, the mutant shows 60% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
K53A | site-directed mutagenesis of the protein part, the mutant shows 80% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
K56A | site-directed mutagenesis of the protein part, the mutant shows 10% increased activity compared to the wild-type enzyme | Thermotoga maritima |
K62A | site-directed mutagenesis of the protein part, the mutant shows unaltered activity compared to the wild-type enzyme | Thermotoga maritima |
K90A | site-directed mutagenesis of the protein part, the mutant shows 70% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
additional information | wild-type and mutant enzyme structure-function analysis, overview. Point mutations in the conserved protein loop (residues 52-57) have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the RNR region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency | Thermotoga maritima |
R14A | site-directed mutagenesis of the protein part, the mutant shows 60% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
R15A | site-directed mutagenesis of the protein part, the mutant shows 70% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
R52A | site-directed mutagenesis of the protein part, the mutant shows 60% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
R59A | site-directed mutagenesis of the protein part, the mutant shows 20% increased activity compared to the wild-type enzyme | Thermotoga maritima |
R60A | site-directed mutagenesis of the protein part, the mutant shows 30% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
R65A | site-directed mutagenesis of the protein part, the mutant shows 50% increased activity compared to the wild-type enzyme | Thermotoga maritima |
R89A | site-directed mutagenesis of the protein part, the mutant shows 94% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
R89A/U52C | site-directed mutagenesis of the protein part (R89A) and the RNA part (U52C), the mutant shows over 99% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
U52C | site-directed mutagenesis of the RNA part, the mutant shows 92% reduced activity compared to the wild-type enzyme | Thermotoga maritima |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten single-turnover reaction kinetics of wild-type and mutant enzymes, overview | Thermotoga maritima |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, the active site includes at least two metal ions, RNA U52 nucleotide binds a metal ion at the active site | Thermotoga maritima |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Thermotoga maritima | the enzyme is an RNA-based enzyme primarily catalyzing 5'-end pre-tRNA processing | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermotoga maritima | Q9X1H4 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
ribonucleoprotein | - |
Thermotoga maritima |
Purification (Comment) | Organism |
---|---|
recombinant GST-tagged wild-type and mutant proteins and RNA from Escherichia coli strain BL21(DE3)pLysS | Thermotoga maritima |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the enzyme is an RNA-based enzyme primarily catalyzing 5'-end pre-tRNA processing | Thermotoga maritima | ? | - |
? | |
pre-tRNAPhe + H2O | - |
Thermotoga maritima | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | wild-type and mutant enzyme structure-function analysis, overview | Thermotoga maritima |
Synonyms | Comment | Organism |
---|---|---|
RNase P | - |
Thermotoga maritima |
General Information | Comment | Organism |
---|---|---|
additional information | wild-type and mutant enzyme structure-function analysis, overview. RNA U52 and two bacterially conserved protein residues, F17 and R89, are essential for efficient Thermotoga maritima enzyme activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned over 20 A from the cleavage site, probably making contacts with N-4 and N-5 nucleotides of the pretRNA 5'-leader | Thermotoga maritima |
physiological function | the enzyme is an RNA-based enzyme primarily responsible for 5'-end pre-tRNA processing | Thermotoga maritima |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.83 | - |
pre-tRNAPhe | recombinant mutant R89A/U52C, pH 7.5, 37°C | Thermotoga maritima | |
1 | - |
pre-tRNAPhe | recombinant mutant F17A/U52C, pH 7.5, 37°C | Thermotoga maritima | |
8.3 | - |
pre-tRNAPhe | recombinant mutant F17A, pH 7.5, 37°C | Thermotoga maritima | |
10 | - |
pre-tRNAPhe | recombinant mutant R89A, pH 7.5, 37°C | Thermotoga maritima | |
13 | - |
pre-tRNAPhe | recombinant mutant U52C, pH 7.5, 37°C | Thermotoga maritima | |
28.3 | - |
pre-tRNAPhe | recombinant mutant F21A, pH 7.5, 37°C | Thermotoga maritima | |
40 | - |
pre-tRNAPhe | recombinant mutant K53A, pH 7.5, 37°C | Thermotoga maritima | |
44.2 | - |
pre-tRNAPhe | recombinant mutant K90A, pH 7.5, 37°C | Thermotoga maritima | |
54.2 | - |
pre-tRNAPhe | recombinant mutant R15A, pH 7.5, 37°C | Thermotoga maritima | |
65 | - |
pre-tRNAPhe | recombinant mutant R14A, pH 7.5, 37°C | Thermotoga maritima | |
66.7 | - |
pre-tRNAPhe | recombinant mutant K52A, pH 7.5, 37°C | Thermotoga maritima | |
70 | - |
pre-tRNAPhe | recombinant mutant K51A, pH 7.5, 37°C | Thermotoga maritima | |
120 | - |
pre-tRNAPhe | recombinant mutant R60A, pH 7.5, 37°C | Thermotoga maritima | |
170 | - |
pre-tRNAPhe | recombinant mutant K62A, pH 7.5, 37°C | Thermotoga maritima | |
180 | - |
pre-tRNAPhe | recombinant wild-type enzyme, pH 7.5, 37°C | Thermotoga maritima | |
181.7 | - |
pre-tRNAPhe | recombinant mutant K56A, pH 7.5, 37°C | Thermotoga maritima | |
198.3 | - |
pre-tRNAPhe | recombinant mutant R59A, pH 7.5, 37°C | Thermotoga maritima | |
261.7 | - |
pre-tRNAPhe | recombinant mutant R65A, pH 7.5, 37°C | Thermotoga maritima |