Mutations | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) |
|---|
Wild type | 0.85 ± 0.12 | 1.08 ± 0.03 | 1.27 ± 0.15 |
D305V | 0.54 ± 0.08 | 1.43 ± 0.03 | 2.65 ± 0.17 |
D305A | 0.46 ± 0.11 | 1.53 ± 0.02 | 3.33 ± 0.21 |
D305G | 0.36 ± 0.14 | 1.70 ± 0.02 | 4.72 ± 0.33 |
- The reaction was carried out in 2 mL volumes containing 0.2 M KP (potassium phosphate) buffer (pH 8.0), 0.8 U/mL purified SMOA, 1.6 U/mL of purified SMOB, 1.7 U/mL formate dehydrogenase (FDH; EC 1.2.1.2, from Candida boidinii), 0.2 M sodium formate, 0.3 mM NADH, 1 mM NAD+, 0.05 mM FAD, and varying concentrations of styrene (from a 200-fold stock in ethanol). All enzymes were previously expressed in E. coli BL21. The reaction mixture was shaken at 200 rpm and 30 °C for 1 h, and then it was extracted with ether and analyzed with reverse phase HPLC on a Luna C18 (4.6 mm × 150 mm) column at a flow rate of 0.8 mL/min
