Valérie Abécassis

Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450

Abstract Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these approaches, two combinatorial libraries of cytochrome P450 variants derived from the CYP1A subfamily were constructed and their sequence diversity characterized. The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204–214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data. Moreover, mutations in this segment do not alter the discrimination between alkoxyresorufins, which, for all tested mutants, remained similar to that of wild-type CYP1A1. In contrast, the constructed CYP1A1–CYP1A2 mosaic structures, containing multiple crossovers, exhibit a wide range of substrate preference and regioselectivity. These mosaic structures also discriminate between closely related alkoxyresorufin substrates. These results open the way to global high-throughput analysis of structure–function relationships using combinatorial libraries of enzymes together with libraries of structurally related substrates.

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

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.