Table 3. Characteristics of wild-type, M9, and M9* ADHA.
Activity measurements were performed at 25 °C in duplicate or triplicate and the respective Michaelis-Menten plots are depicted in Figure 6. Melting curves are depicted in Figure 2—figure supplement 1. Conversions were performed with 5 µM of ADHA and 10 mM of prochiral ketone substrate: ethyl 4-chloro-3-oxobutanoate (COBA) and 4-chloroacetophenone (4-CAP). Details and chromatograms are provided in Supplementary file 4.
| Enzyme |
Tm
(°C) |
kcat
(s−1) |
KM,NADP+
(µM) |
kcat/KM,NADP+
(s−1 mM−1) |
Conversion and ee (COBA) | Conversion and ee (4-CAP) |
|---|---|---|---|---|---|---|
| ADHA WT | 43.0 ± 0 | 1.1 ± 0.04 | 9.5 ± 1.2 | 116 ± 35 | > 99% > 99% ee (R) |
> 99% 97.2% ee (S) |
| M9 | 94.5 ± 0 | 0.27 ± 0.02 | 1040 ± 108 | 0.26 ± 0.2 | n.d. | n.d. |
| M9* | 88.0 ± 0 | 0.7 ± 0.01 | 9.6 ± 0.7 | 73 ± 14.5 | > 99% > 99% ee (R) |
> 99% 98% ee (S) |