Table 3.
Phosphatase activities of PgpB variants.
| Protein | % of wt PgpB activity | |
|---|---|---|
| C55-PPa | PGPa | |
| PgpB | 100 ± 16 (730 ± 120 nmol min−1 mg−1) | 100 ± 19 (3,300 ± 630 nmol min−1 mg−1) |
| C1motif | ||
| K97A | 118 ± 12 | 298 ± 55 |
| ΔV100 | 2.7 ± 0.6 | 0.4 ± 0.1 |
| R104A | 2.0 ± 0.4 | 0.4 ± 0.1 |
| P105A | 3 ± 1 | 0.4 ± 0.3 |
| C2motif | ||
| E154A | 62 ± 11 | 61 ± 5 |
| P160A | 13 ± 2 | 2.5 ± 0.1 |
| S161A | 0.14 ± 0.08 | 0.03 ± 0.01 |
| G162A | 0.8 ± 0.1 | 0.2 ± 0.04 |
| G162D | NDb | 0.03 ± 0.02 |
| H163A | 27 ± 3 | 0.09 ± 0.03 |
| E154A/H163A | 10 ± 2 | 0.03 ± 0.01 |
| C3motif | ||
| S200A | 55 ± 7 | 162 ± 23 |
| R201A | 25 ± 6 | 0.3 ± 0.1 |
| H207A | ND | ND |
| W208A | 18 ± 4 | 24 ± 2 |
| D211A | 0.07 ± 0.02 | 0.1 ± 0.1 |
aThe enzymatic activity was measured in the presence of 50 µM of [14C]C55-PP or [14C]PGP substrate and an appropriate amount of enzyme to obtain less than 30% of hydrolysis. The product and substrate were separated by TLC and subsequently quantified by radioactivity counting.
bND no detectable activity with up to 2 µg of pure protein.