Received for publication June 2, 2008.
Revised August 12, 2008.
Accepted for publication August 13, 2008.

The Application of Molecular Modeling for Prediction of Substrate Specificity in Cytochrome P450 1A2 Mutants

Abstract

Molecular dynamics (MD) simulations of 7-ethoxy and 7-methoxyresorufin bound in the active site of P450 1A2 wild type and various mutants were used to predict changes in substrate specificity of the mutants. A total of 26 multiple mutants representing all possible combinations of five key amino acid residues which are different between P450 1A1 and 1A2, were examined. The resorufin substrates were docked in the active site of each enzyme in the productive binding orientation and MD simulations were performed on the ES complex. Ensembles collected from MD trajectories were then scored based on geometric parameters relating substrate position with respect to the activated oxoheme cofactor. The results showed a high correlation between the previous experimental data on P450 1A2 wild type and single mutants with respect to the ratio between 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) activities, and the equivalent in silico E/M scores. Moreover, this correlation served to establish linear regression models utilized to evaluate E/M scores of multiple P450 1A2 mutants. Seven mutants, all of them incorporating the L382V substitution, were predicted to shift specificity to that of P450 1A1. The predictions were then verified experimentally. The appropriate P450 1A2 multiple mutants were constructed by site-directed mutagenesis, expressed in E. coli, and assayed for EROD and MROD activities. Out of six mutants, five demonstrated increased EROD/MROD ratio confirming modeling predictions.

Key words: computational models, computer modeling and simulation, CYP1A, cytochrome P450, cytochrome P450 catalyzed oxidations, cytochrome P450 function, cytochrome P450 isoforms, ligand docking, structure-activity relationships