The human cytochrome P450 2A family: Comparisons and identification of amino acids essential for substrate recognition
DeVore, Natasha M.
University of Kansas
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The goal of this research was to identify the differential structure-activity relationships between cytochromes P450 (CYP) 2A13 and 2A6 and their substrates. Cytochromes P450 2A13 and 2A6 are very closely related, having 94% amino acid sequence identity. Both proteins metabolize drugs, toxins, and procarcinogens, including the nicotine derivative 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1. Yet CYP2A13 has a much higher rate of metabolism than CYP2A6 for both compounds. Combined with its increased affinity and expression in the respiratory tract, CYP2A13 is a candidate for breakdown of NNK into the reactive metabolites that can form DNA adducts in the lung, leading to the development of lung cancer (1). Thus, understanding differential metabolism is important. Due to the high identity of CYP2A13 and CYP2A6, we hypothesized that a few amino acids found within the active site are responsible for CYP2A substrate specificity. Of the 32 amino acids which differ between CYP2A13 and CYP2A6, ten are located in regions of the protein that often determine substrate recognition in cytochromes P450 (2). Crystallization of CYP2A13 by the Scott lab verified that these amino acids were in or near the active site. The goal of this project was to determine which of these ten amino acids were vital for substrate selectivity in the CYP2A enzymes. Several different methods were employed throughout this study including site-directed mutagenesis, ligand binding assays, metabolism assays, molecular modeling, and crystallography. Site-directed mutagenesis was used to construct ten single residue CYP2A13 mutants with each mutation substituting the residue found in CYP2A6 in that position. The consequences of these ten mutations to the binding of coumarin, 2′-methoxyacetophenone, phenyl isothiocyanate, and phenacetin were determined using spectral ligand binding assays. The amino acids in positions 117, 208, 300, 301, 365, and 369 caused significant changes in ligand affinity. To determine if these amino acids were also essential for substrate metabolism, phenacetin was used as a structural probe since CYP2A6 converts phenacetin to acetaminophen with decreased catalytic efficiency compared to CYP2A13 (3). Employing this assay, four mutations (S208I, F300I, A301G, and G369S) were identified that diminished CYP2A13 phenacetin O-deethylation to near CYP2A6 activity. Two mutants, CYP2A13 A117V and L366I, both increased enzyme activity more than four-fold. Construction of the reverse mutant series, a CYP2A6 protein incorporating the residues found at the corresponding active site positions in CYP2A13, confirmed that positions 208, 300, 301, and 369 were jointly responsible for phenacetin metabolism and binding. While all single 2A6 mutants had very low-level activity, the double, triple, and a quadruple mutant with changes at these four positions increasingly conferred phenacetin metabolism to CYP2A6. A structural basis for the effects of these four mutations on phenacetin binding and metabolism was explored with molecular docking studies using Surflex-Dock (4). The results suggested that the ability of CYP2A13 to bind and metabolize phenacetin was due to steric variations among key residues in the CYP2A13 and CYP2A6 active sites. This was confirmed with the crystal structure of the CYP2A6 I208S/I300F/G301A/G369S complexed with phenacetin. Finally, this study characterized the effects of naturally occurring CYP2A13 polymorphisms on the binding of the well-known CYP2A ligand coumarin (5). All of these polymorphisms were located on the exterior portions of the protein. None of the polymorphisms examined resulted in a significant change in ligand affinity, indicating that having one of these variants would not substantially alter an individual's ability to metabolize CYP2A13 substrates. Thus, the research presented in this thesis provides structural and functional insight into CYP2A13 and CYP2A6 substrate selectivity, which is largely modulated by the steric effects mediated by the differential amino acids at positions 208, 300, 301, and 369. References 1. Su, T., Bao, Z. P., Zhang, Q. Y., Smith, T. J., Hong, J. Y., and Ding, X. X. (2000) Cancer Res. 60(18), 5074-5079 2. Zhang, J. Y., Wang, Y., and Prakash, C. (2006) Curr Drug Metab 7(8), 939-948 3. Bieche, I., Narjoz, C., Asselah, T., Vacher, S., Marcellin, P., Lidereau, R., Beaune, P., and de Waziers, I. (2007) Pharmacogenetics and Genomics 17(9), 731-742 4. Nishimura M Fau - Yaguti, H., Yaguti H Fau - Yoshitsugu, H., Yoshitsugu H Fau - Naito, S., Naito S Fau - Satoh, T., and Satoh, T. (0031-6903 (Print)) 5. Schlicht, K. E., Michno, N., Smith, B. D., Scott, E. E., and Murphy, S. E. (2007) Xenobiotica, 1-11
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