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

Background: High-throughput (HT) characterization of drugs for potential biotransformation and interaction is routine in pharmaceutical industry. Objective: HT approaches were extended to enzyme studies for identifying combinations of structural elements that control substrate specificity. Methods: 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. Results/conclusion: 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.