TABLE 3.
Kinetic properties of recombinant poplar DQD/SDH (Poptr1 and Poptr5) and QDH (Poptr2 and Poptr3)
Either shikimate or quinate was used as a substrate with at least 12 concentrations of substrate (ranging from 0.5 to 2000 μm). The tested cofactor (either NADP+ or NAD+) was kept at saturating concentrations. Vmax and Km values were estimated based on nonlinear fitting to the Michaelis-Menten model using the Curve Fitting ToolboxTM implemented in MATLAB. Vmax and Km values represent averages of three independent protein purifications. Confidence of curve fitting (i.e. 95% confidence bounds) was assessed with MATLAB. Confidence bounds are indicated as ± of the respective averages. Substrate specificities were calculates by dividing Vmax by Km.
| Substrate tested | Cofactor used | Vmax | Km | Vmax/Km | |||
|---|---|---|---|---|---|---|---|
| μm s−1 mg−1 | μm | ||||||
| Poptr1 | Shikimate | NADP+ | 323.1 | ± 11.2 | 223.1 | ± 24.4 | 1.45 |
| Shikimate | NAD+ | 17.8 | ± 3.2 | 426.9 | ± 243.8 | 0.04 | |
| Quinate | NADP+ | NDa | |||||
| Quinate | NAD+ | ND | |||||
| Poptr2 | Shikimate | NADP+ | ND | ||||
| Shikimate | NAD+ | 3.1 | ± 0.5 | 833.1 | ± 382.9 | 0.003 | |
| Quinate | NADP+ | ND | |||||
| Quinate | NAD+ | 67.0 | ± 3.5 | 334.4 | ± 47.1 | 0.20 | |
| Poptr3 | Shikimate | NADP+ | ND | ||||
| Shikimate | NAD+ | 18.0 | ± 1.4 | 391.8 | ± 92.3 | 0.05 | |
| Quinate | NADP+ | ND | |||||
| Quinate | NAD+ | 73.8 | ± 4.7 | 321.3 | ± 55.3 | 0.23 | |
| Poptr5 | Shikimate | NADP+ | 70.0 | ± 9.0 | 345.8 | ± 96.2 | 0.20 |
| Shikimate | NAD+ | ND | |||||
| Quinate | NADP+ | ND | |||||
| Quinate | NAD+ | ND | |||||
a ND means no activity detectable or too low to determine kinetic properties.