Design and characterization of a novel "family-shuffling" technology adapted to membrane enzyme: application to P450s involved in xenobiotic metabolism.

Abstract

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