Kanamycin nucleotidyltransferase from Staphylococcus aureus
Enzyme Description
Sequence
MNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTTFLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF
Hans H. Liao et al. (1993)
β Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents
Enzyme and Microbial Technology
Β· doi:10.1016/0141-0229(93)90151-Q β
Β· Activity - Classical Activity + Stability - Incubation
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Database ID
UniProt: P05057 β
Sequence Annotation
Inferred - from protein name
Protein Source
Recombinant, host bacterium Escherichia coli BL21 (DE3)
Experimental Data (48 measurements)
48 measurements β page 1 of 3
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 4% | 100.0 | β | 101.6 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 8% | 100.0 | β | 98.1 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 12% | 100.0 | β | 33.2 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 16% | 100.0 | β | 4.3 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 20% | 100.0 | β | 0.8 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Dimethylformamide (DMF) | 24% | 100.0 | β | 7.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Dimethylformamide (DMF) | 18% | 100.0 | β | 38.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | Inferred - from protein name (2 mutations) | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 100.0 | β | 2.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Ethanol | 15% | 100.0 | β | 80.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Ethanol | 20% | 100.0 | β | 57.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Ethanol | 25% | 100.0 | β | 4.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| WT | Activity + Stability - Incubation | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Ethanol | 30% | 100.0 | β | 0.0 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 20% | 100.0 | 0.8 | 14.5 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| D80Y/T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 20% | 100.0 | 0.8 | 69.2 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 4% | 100.0 | 101.6 | 120.8 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 8% | 100.0 | 98.1 | 98.1 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 12% | 100.0 | 33.2 | 87.2 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| T130K | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 16% | 100.0 | 4.3 | 43.4 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| D80Y | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 4% | 100.0 | 101.6 | 106.3 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
| D80Y | Activity - Classical | Activity after incubation (15 minutes at 22-24Β°C), measured by radioactivity assay (radioactivity measurement of [Β³H]AMP-kanamycin binding to phosphocellulose paper) in the presence of organic solvent | Tetrahydrofuran (THF) | 8% | 100.0 | 98.1 | 102.8 | % | ? | ? | Incubation: 22-24Β°C, Assay: 37Β°C | ? mM AMP-kanamycin, ? mM ATP (Adenosine triphosphate) | AMPβkanamycin , Pyrophosphate | β | β | Non-incubated control (in %), assay in the presence of organic solvent | Clear about assay in the presence of organic solvent |
Mutations in this dataset (4)
WT
D80Y
D80Y/T130K
T130K
Visualization : Activity β Classical
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Visualization : Activity + Stability β Incubation
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.