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.


CONCLUSIONS
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.
Urban
Philippe
P
Centre de Génétique Moléculaire, Laboratoire d'Ingénierie des Protéines Membranaires, CNRS UPR2167, Avenue de la terrasse, 91190 Gif-sur-Yvette, France.
Truan
Gilles
G
Pompon
Denis
D
D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation; D020650:Combinatorial Chemistry Techniques; D003198:Computer Simulation; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1