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Laboratory Evolution of P450 BM‐3 for Mediated Electron Transfer
Author(s) -
Nazor Jovana,
Schwaneberg Ulrich
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500436
Subject(s) - monooxygenase , cofactor , saturated mutagenesis , chemistry , enzyme kinetics , nad+ kinase , electron transfer , cytochrome p450 , mutagenesis , mutant , stereochemistry , enzyme , biochemistry , active site , photochemistry , gene
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 μ M −1 min −1 for P450 BM‐3(F87A R47F) and 34 μ 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 m M Co III sep) to 197 min −1 (0.5 m M Co III sep), and K m decreases simultaneously from 7.0 μ M to 3.7 μ M , for P450 BM‐3 (F87A R47F). For P450 BM‐3 (F87A R47Y), k cat increases from 138 min −1 (0.25 m M Co III sep) up to 187 min −1 (0.5 m M Co III sep), and K m decreases from 8.2 μ M to 4.2 μ M . 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.