TABLE 2.
Biochemical parameters of mFMO and mutant variants
| Enzyme | Mean value ± SD or as indicated |
||||||
|---|---|---|---|---|---|---|---|
| Tm ± 95% CI (°C)a | T50 (°C)b | Topt (°C)c | Optimal pHd | Km (μM)e | kcat (s−1)e | kcat/Km (μM−1 s−1)e | |
| mFMO | 46.2 ± 0.2 | 40.6 ± 0.4 | 40 | 8.5 | 1.07 ± 0.13 | 1.28 ± 0.03 | 1.20 ± 0.12 |
| mFMO_8 | 51.2 ± 0.1 | 45.1 ± 0.9 | 7.5 | ||||
| mFMO_11 | 51.7 ± 0.1 | 47.1 ± 1.2 | 7.5 | ||||
| mFMO_14 | 53.9 ± 0.2 | 49.0 ± 1.5 | 8.0 | ||||
| mFMO_15 | 54.5 ± 0.1 | 49.6 ± 0.4 | 7.5 | ||||
| mFMO_20 | 55.2 ± 0.1 | 50.0 ± 0.4 | 40 | 8.0 | 0.83 ± 0.02 | 0.93 ± 0.05 | 1.11 ± 0.03 |
| mFMO_24 | 52.0 ± 0.1 | 49.2 ± 0.2 | 7.5 | ||||
| mFMO_28 | 50.9 ± 0.1 | 48.9 ± 0.5 | 8.0 | ||||
Melting curves were obtained by CD at pH 7.5, and melting temperature was estimated by four-parameter logistic regression of the melting curve.
The temperature where half of the enzyme activity was lost (T50) was measured at pH 7.5, and the reported data are the mean values ± standard deviations (SD) from three independent experiments.
Optimal temperature (Topt) was determined at pH 8.0 for mFMO and mFMO_20.
Optimal pH for TMA conversion was determined at 22°C.
Steady-state kinetic measurements were performed with various concentrations of TMA (Sigma-Aldrich) as the substrate and a fixed NADPH concentration (200 μM) at 23°C, pH 8.0. Reported data are the mean values ± SD from two biological replicates.