The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism

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Abstract

Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382  Ala, were constructed, expressed in Escherichia coli, and purified by Ni–NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.

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Materials

The DNA sequencing and mutagenic oligonucleotide primers were synthesized at the Molecular Genetics Instrumentation Facility, University of Georgia, Athens, GA. Primers for the construction of the expression construct were synthesized by Bethesda Research Laboratories (Gaithersburg, MD). Restriction endonucleases, the GeneEditor in vitro site-directed mutagenesis system, R408 helper phage, E. coli DH5α competent cells, and plasmid preparation kit were obtained from Promega (Madison, WI).

Expression and purification of P450 1A1 and 1A2 WT and mutant enzymes

The choice of P450 1A1 and 1A2 residues subjected to mutagenesis was based on the analysis of enzyme–substrate interactions in the active site of the P450 1A1 homology model and the sequence alignment between 1A1 and 1A2. Among several key active site residues identified [10], only five were different between P450 1A1 and 1A2. The positions of these residues in a linear sequence are shown in Fig. 2A. In P450 1A1, the unique active site residues are thus Ser-122, Asn-221, Gly-225, Leu-312, and

Discussion

The overall objective of this study was to evaluate sites suggested by the predictions from homology modeling and substrate docking, and to alter substrate specificity of the P450 1A enzymes by single reciprocal mutations. Additionally, we examined the utility of molecular modeling to provide rationale for substrate specificity in terms of substrate dynamics in the active site. In our previous work [10], we have shown that binding free energy calculations in homology models can be successfully

Acknowledgements

This work was supported by NIH Grants CA85542 and RR16440 (G.D.S.) and by ES07628 (C.W.F.). Modeling studies were performed at the Computational Chemistry and Molecular Modeling Laboratory, Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV.

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