High-throughput enzymology and combinatorial mutagenesis for mining cytochrome P450 functions

High-throughput (HT) characterization of drugs for potential biotransformation and interaction is routine in pharmaceutical industry.

HT approaches were extended to enzyme studies for identifying combinations of structural elements that control substrate specificity.

Structure-based and combinatorial mutagenesis have been applied with success to decipher P450 structure-function relationships. The idea is to measure activities on a library of combinatorial variants of similar structure with a large collection of substrates presenting a similar chemical scaffold. This combinatorial approach is then associated to multivariate statistics to relate functional features to structural determinants.

A method to measure HT kinetics is presented. The proposed statistical approach is illustrated with tri- and tetracyclic substrates and artificial variant enzymes of the CYP1A subfamily.