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

Abstract

ABSTRACT:Molecular dynamics (MD) simulations of 7-ethoxy-and 7-methoxyresorufin bound in the active site of cytochrome P450 (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 enzyme-substrate complex. Ensembles collected from MD trajectories were then scored on the basis of 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" (the ratio of hits obtained with 7-ethoxyresorufin to those obtained with 7-methoxyresorufin). Moreover, this correlation served to establish linear regression models used 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 Escherichia coli, and assayed for EROD and MROD activities. Of six mutants, five demonstrated an increased EROD/MROD ratio, confirming modeling predictions.Cytochromes P450 constitute a large family of heme-thiolate monooxygenase enzymes widely found in nature (Nelson et al., 1996). These enzymes are capable of catalyzing the oxidation of a wide variety of both xenobiotic and endogenous compounds. However, even closely related isoforms may exhibit different catalytic activities.Human P450 1A1 and 1A2, the two major isoforms in the P450 1A subfamily, share 72% amino acid sequence identity but display different substrate specificities. P450 1A1 prefers to metabolize benzo- [a]pyrene and other polycyclic aromatic hydrocarbons, whereas P450 1A2 favors the oxidation of heterocyclic and aromatic amines (Kawajiri and Hayashi, 1996;Guengerich, 2005). Likewise, they also exhibit different substrate specificities with resorufin substrates such as 7-ethoxyresorufin and 7-methoxyresorufin (Nerurkar et al., 1993;Burke et al., 1994). Therefore, the P450 1A1/1A2 system provides a good model for exploring the basis for functional differences between individual P450 enzymes.The experimentally determined structure of a protein can provide valuable insight into its function. Homology modeling is an alternative method for obtaining the structure when the crystal structure is not available (Szklarz et al., 2000). Molecular dynamics (MD) simulations on the enzyme-substrate complex could provide information on whether the substrat...

Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with Isoform-Selective Metabolism

“…The overall yield of purified enzyme was approximately 20 to 40%, similar to that reported previously (Liu et al, 2004;Tu et al, 2008;Huang and Szklarz, 2010). The purity of CYP1A1 and CYP1A2 verified by SDS-polyacrylamide gel electrophoresis and Western blots indicated that the proteins were at least 95% pure.…”

“…The differences in activities probably arise from structural differences between CYP1A1 and CYP1A2. Our previous studies indicated that five key residues in the active site that differ between the enzymes play a role in the determination of substrate specificity with alkoxyresorufins (Liu et al, 2003(Liu et al, , 2004Tu et al, 2008) and phenacetin (Huang and Szklarz, 2010). In CYP1A1, the unique active site residues are Ser122, Asn221, Gly225, Leu312, and Val382, which correspond to Thr124, Thr223, Val227, Asn312, and Leu382 of CYP1A2, respectively.…”

“…The enzymes were expressed and purified as described previously (Liu et al, 2003(Liu et al, , 2004Tu et al, 2008;Huang and Szklarz, 2010). In brief, His-tag-containing CYP1A1 and CYP1A2 were expressed in Escherichia coli DH5␣ cells, and the enzymes were purified by affinity chromatography with nickel-nitrilotriacetic acid agarose.…”

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

“…The resulting trajectory was analyzed to find the best substrate orientation, and the enzymesubstrate complex was minimized for another 1000 steps, as described earlier. This optimal substrate orientation was used as a starting point for automated docking with the Affinity module of Insight II using default parameters, as described earlier (Ericksen and Szklarz, 2005;Tu et al, 2008;Huang and Szklarz, 2010;Walsh et al, 2013). Residues within 10 Å from the initial substrate position comprised the flexible region of the protein during all docking runs.…”

Significant Increase in Phenacetin Oxidation on L382V Substitution in Human Cytochrome P450 1A2

“…Moreover, mutations at position 382 in both CYP1A1 and CYP1A2 shifted substrate specificity from one enzyme to another (Liu et al, 2004). Further computational and experimental studies with multiple CYP1A2 mutants confirmed the importance of this residue for alkoxyresorufin oxidation (Tu et al, 2008). Therefore, it would be of interest to examine the effect of these mutations on oxidation of other types of substrates.…”