Laboratory evolution of P450BM-3 for mediated electron transfer

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 mu M-1 min(-1) for P450 BM-3(F87A R47F) and 34 mu M-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 mu M to 3.7 mu M, 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 mu M to 4.2 mu M. Due to lower K. 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.