Design and Characterization of a Novel « Family-Shuffling » Technology Adapted to Membrane Enzyme: Application to P450s Involved in Xenobiotic Metabolism

摘要: Cytochrome P450 functional diversity and their predominant role in drug pollutant metabolism toxicity1 makes these enzymes particularly suitable for the design of new catalysts as well structure-function analysis2. Combinatorial molecular evolution (CME) is a powerful approach used tuning protein functions3;4and investigation biochemical mechanisms driving substrate recognition5 or catalysis6. Family-shuffling has proved to accelerate process7. A low content mosaic structures was frequently reported libraries constructed using DNase I fragmentation8. We designed strategy family shuffling yeast expression vectors. This procedure takes advantage association between vitro 9 vivo 10. recombination build high complexity library containing levels parental structures. The use engineered strains membrane proteins into an optimized redox environment 11 also allows efficient bioconversion. model human CYPIAI CYPIA2 which share 71% identity have distinct, while overlapping, specificities.