TABLE 3.
Kinetic analysis of OHB reductases Ec.Mdh5Q and Ec.Mdh7Q.
| Enzyme | Ec.Mdh5Q | Ec.Mdh7Q |
|---|---|---|
| NADH a | ||
| Vmax,app (U mg−1) | 67.68 (±3.12) | 20.02 (±1.99) |
| kcat,app (s−1) | 36.48 (±1.68) | 10.79 (±1.07) |
| Km,app (mM) | 0.04 (±0.004) | 0.27 (±0.04) |
| kcat,app/Km,app (mM−1 s−1) | 1,027.40 (±67.46) | 40.35 (±1.71) |
| NADPH a | ||
| Vmax,app (U mg−1) | 5.56 (±0.64) | 118.80 (±18.5) |
| kcat,app (s−1) | 2.99 (±0.35) | 64.03 (±9.97) |
| Km,app (mM) | 0.32 (±0.04) | 0.10 (±0.03) |
| kcat,app/Km,app (mM−1 s−1) | 9.50 (±0.05) | 645.47 (±76.25) |
| OHB b | ||
| Vmax,app (U mg−1) | 138.50 (±10.45) | 154.80 (±6.30) |
| kcat,app (s−1) | 74.89 (±5.63) | 83.43 (±3.40) |
| Km,app (mM) | 1.84 (±0.50) | 1.08 (±0.19) |
| Ki (mM) | 31.9 (±5.6) c | 5.45 (±0.48) |
| kcat,app/Km,app (mM−1 s−1) | 44.91 (±15.18) | 80.29 (±17.27) |
| Specificity | ||
| (kcat,app/Km,app)NADPH/(kcat,app/Km,app)NADH | 0.01 | 16.00 |
| Overall catalytic efficiency | ||
| (kcat,app/Km,app)OHB × (kcat,app/Km,app)NAD(P)H | 46,136 | 51,823 |
Specific OHB reductase activities were determined at fixed concentrations of substrate (OHB, 2 mM) and variable concentrations of NAD(P)H (1–0.03 mM).
Specific OHB reductase activities were determined at fixed concentrations (0.25 mM) of the preferred co-substrate NAD(P)H and variable amounts of OHB (10–0.005 mM).
Apparent kinetic constants (Km,app, Vmax,app) were estimated by fitting the experimental data to the Michaelis–Menten model using non-linear regression unless enzymes displayed substrate inhibition kinetics (Curve fitting tool, MATLAB R2021a). To calculate the apparent catalytic constant kcat,app, the molecular weight of one subunit of Ec.Mdh (32.337 kDa) was considered. Specificity and efficiency were calculated based on mean kcat,app and Km,app values. Enzyme assays were performed in biological duplicates. Ec.Mdh5Q contains mutations I12V:R81A:M85Q:D86S:G179D.
Ec.Mdh7Q additionally contains mutations D34G:I35R.