title: Regioselectivity of CYP2B6: homology modeling, molecular dynamics simulation, docking.
abstract: Human cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifosfamide (IFA), thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modeled based on the structure of rabbit CYP2C5. We modeled the missing 22-residue loop in CYP2C5 between helices F and G (the F-G loop), which is not resolved in the X-ray structure, by molecular dynamics (MD) simulations using a simulated annealing protocol. The modeled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 A between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydrophobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets were compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site, both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates and the enzyme.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Bathelt
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pleiss
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D001665:Binding Sites; D003198:Computer Simulation; D003520:Cyclophosphamide / Q000737:chemistry; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D000072467:Cytochrome P450 Family 2; D006801:Humans; D007069:Ifosfamide / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry*; D011817:Rabbits; D016415:Sequence Alignment; D013255:Steroid 21-Hydroxylase / Q000737:chemistry; D040681:Structural Homology, Protein
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; C065407:CYP2C5 protein, Oryctolagus cuniculus; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase; D010089:Oxidoreductases, N-Demethylating; D007069:Ifosfamide