Abstract
Preparative synthesis with P450 monooxygenases is hampered in cell‐free systems by the requirement for cofactors such as NAD(P)H as reduction equivalents. A validated medium‐throughput screening system was designed for improving P450 monooxygenases by mediated electron transfer with zinc/cobalt(III)sepulchrate (Zn/Co^III^sep) as an alternative and cost‐effective cofactor system. The monooxygenase P450 BM‐3 F87A was used as a model system for developing the screening system in a 96‐well format. A coefficient of variation of less than 10 % was achieved under optimized screening conditions. The mediator evolution screen was validated by comparing the activity of P450 BM‐3 to P450 BM‐3 F87A and by screening a saturation mutagenesis library at amino acid position R47. For mediated electron transfer, two double mutants P450 BM‐3(F87A R47F) and P450 BM‐3 (F87A R47Y) were identified with a two–threefold increased catalytic efficiency (up to 32 μM^−1^min^−1^ for P450 BM‐3(F87A R47F) and 34 μM^−1^min^−1^ for P450 BM‐3 (F87A R47Y)) compared to P450 BM‐3 F87A. The kinetic constants of the double mutants are, in contrast to those of P450 BM‐3 F87A, dependent on Co^III^sep concentration in the presence of NADPH. k~cat~ increases from 145 min^−1^ (0.25 mM Co^III^sep) to 197 min^−1^ (0.5 mM Co^III^sep), and K~m~ decreases simultaneously from 7.0 μM to 3.7 μM, for P450 BM‐3 (F87A R47F). For P450 BM‐3 (F87A R47Y), k~cat~ increases from 138 min^−1^ (0.25 mM Co^III^sep) up to 187 min^−1^ (0.5 mM Co^III^sep), and K~m~ decreases from 8.2 μM to 4.2 μM. Due to lower K~m~ values, the catalytic efficiencies were improved six times for P450 BM‐3 (F87A R47F) and three times for P450 BM‐3 (F87A R47Y), when comparing catalytic efficiencies of the mediated electron‐transfer system to the natural reduction equivalent NADPH.