Table 1. SsoPox lactonase activity characterization.
|
Substrate
|
wild-type SsoPox
|
|||||
|---|---|---|---|---|---|---|
| Name | Additive | kcat (s-1) | KM (µM) | KI (µM) | kcat/KM (s -1 M-1) | |
| AHLs | C4-AHL (r) (V) | - | ND | ND | ND | 11.62 ± 0.72 |
| C6-AHL (r) (VI) | - | 0.36 ± 0.01 | 459 ± 49 | - | 7.84 (±0.87)x102 | |
| C8-AHL (r) (VII) | - | 1.00 ± 0.04 | 145 ± 26 | - | 6.90 (±1.27)x103 | |
| C12-AHL (r) (VIII) | - | 1.70 ± 0.31 | 345 ± 90 | - | 4.93 (±1.56)x102 | |
| 3-oxo-AHLs | 3-oxo-C6-AHL (l) (IX) | - | 0.083 ± 0.003 | 558 ± 68 | - | 1.49 (±0.20)x102 |
| 3-oxo-C6-AHL (r) (IX) | - | 0.041 ± 0.005 | 592 ± 176 | - | 6.87 (±2.20)x101 | |
| 3-oxo-C8-AHL (l) (X) | - | 0.54 ± 0.02 | 123 ± 22 | - | 4.39 (±0.80)x103 | |
| 3-oxo-C8-AHL (r) (X) | - | 0.42 ± 0.02 | 256 ± 55 | - | 1.63 (±0.36)x103 | |
| 3-oxo-C10-AHL (l) (XI) | - | 4.52 ± 0.10 | 143 ± 15 | - | 3.16 (±0.33)x104 | |
| + 0.1% SDS | ND | ND | - | 1.96 (±0.04)x102 | ||
| + 0.01% SDS | 0.75 ±0.03 | 243±43 | - | 3.09 (±0.56)x103 | ||
| 3-oxo-C12-AHL (l) (XII) | - | 1.01 ± 0.13 | 456 ± 128 | - | 2.22 (±0.68)x103 | |
| γ-lactones | γ-butyrolactone (XIII) | - | ND | ND | ND | 1.20 (±0.12)x103 |
| γ-heptanolide (r) (XIV) | - | 2.92 ± 0.08 | 166 ± 21 | - | 1.76 (±0.23)x104 | |
| Nonanoic-γ-lactone (r) (XV) | - | 5.54 ± 0.57 | 2 943 ± 436 | - | 1.88 (±0.34)x103 | |
| Undecanoic-γ-lactone (r) (XVI) | - | 4.95 ± 0.26 | 2 099 ± 230 | - | 2.36 (±0.29)x103 | |
| + 0.1% SDS | 2.23 ± 0.47 | 1 250 ± 361 | 1 470 ± 440 | 1.78 (±0.64)x103 | ||
| + 0.01% SDS | 0.46 ± 0.01 | 94 ± 10 | - | 4.89 (± 0.53)x103 | ||
| Dodecanoic-γ-lactone (r) (XVII) | - | 2.72 ± 0.13 | 1 220 ± 144 | - | 2.23 (± 0.28)x103 | |
| δ-lactones | δ-valerolactone (XVIII) | - | ND | ND | ND | ND |
| Nonanoic-δ-lactone (r) (XIX) | - | 15.32 ± 0.52 | 359 ± 63 | - | 4.27 (±0.77)x104 | |
| Undecanoic-δ-lactone (r) (XX) | - | 7.38 ± 0.28 | 94 ± 18 | - | 7.86 (±1.53)x104 | |
| Dodecanoic-δ-lactone (r) (XXI) | - | 12.65 ± 0.44 | 1 678 ± 133 | - | 7.54 (±0.65)x103 | |
| Others | ε-caprolactone (XXII) | - | 4.45 ± 0.08 | 234 ± 18 | - | 1.90 (±0.15)x104 |
| Dihydrocoumarin (XXIII) | - | 7.32 ± 1.25 | 1 376 ± 455 | - | 5.32 (±1.98)x103 | |
Roman numbers indicate the chemical structures of indicated molecules presented in Figure S1 . r corresponds to racemic solution and l at the pure levorotatory enantiomer. Data obtained with cobalt as cofactor. ND corresponds to an undetermined value. When Vmax could not be reached, the linear part of the MM plot was fitted to a linear regression and corresponded to the catalytic efficiency. SsoPox clearly preferred the levorotatory enantiomer of the AHLs; kinetics performed for the racemic mix resulted in characterization for the levorotatory enantiomer.