Table 1. Summary of structural and functional data for ADNV endonuclease mutants and hypothetical role of mutated residues.
| Mutation | ANDV Lfull | ANDV L1–200 | |||
|---|---|---|---|---|---|
| Expression in mammalian cells 1 | Expression in bacteria 2 | Thermal stability 3 | Stabilization by Mn2+ 4 | Ribonuclease activity 5 | |
| Wild-type | |||||
| None | – | – | NA | NA | NA |
| Active site residues involved in catalysis and Mn 2+ coordination | |||||
| H36R | +++ | ++ | 43°C | + 5°C | 0 |
| D97E | +++ | +++ | 47°C | – | 0 |
| E110A | +++ | ++ | 45°C | + 7°C | 0 |
| Active site residues positioning catalytic residues or substrate | |||||
| D37A | + | – | NA | NA | NA |
| P96A | +++ | +++ | 45°C | + 10°C | 14 |
| K124A | +++ | ++ | 49°C | + 12°C | 2 |
| K127A | ++ | ++ | 46°C | + 12°C | 15 |
| Residues contributing to substrate binding outside the active site | |||||
| Y32V | ++ | – | NA | NA | NA |
| R35H | +++ | +++ | 45°C | + 11°C | 20 |
| Residues with stabilizing contacts near the active site | |||||
| D40E | +++ | + | 37°C | + 11°C | 96 |
| I43A | +++ | + | 34°C | + 10°C | 83 |
| K44A | +++ | + | 38°C | + 13°C | 75 |
| N50A | + | +++ | 41°C | + 12°C | 22 |
| N98A | + | + | 43°C | + 11°C | 83 |
| Residue positioning helix e | |||||
| N167A | + | + | 38°C | + 13°C | 100 |
1 ANDV full-length L protein expression in mammalian cells upon transient transfection. The steady-state protein level was estimated from immunoblot signals shown by Heinemann et al. [37].
2 Semiquantitative representation of protein yield from + to +++. A “–”denotes apparent toxicity of the expressed protein preventing growth of transformed bacteria. A reduced growth rate was observed with bacteria expressing L1–200 N167A protein.
3 Melting temperatures measured by thermofluor assay.
4 Increase in melting temperature upon the addition of 16 mM MnCl2 measured by thermofluor assay.
5 Measured by ribonuclease assay. The percentage of degraded substrate RNA after 2 h of incubation is given in %.
NA, not applicable.