A Model for Human Cytochrome P₄₅₀ 2D6 Based on Homology Modeling and NMR Studies of Substrate Binding

Abstract

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.

Characterization of Cytochrome P450 2D6.1 (CYP2D6.1), CYP2D6.2, and CYP2D6.17 Activities toward Model CYP2D6 Substrates Dextromethorphan, Bufuralol, and Debrisoquine

“…In the presence of all three amino acid substitutions, apparent K m for both bufuralol and codeine was 5-to 10-fold higher than that observed for the CYP2D6.1 protein. Data from structural models and sequence alignment data indicate that Thr-107 and Arg-296 lie within distinct substrate contacting regions of the CYP2D6 active site (Hasemann et al, 1995;Modi et al, 1996). Consistent with the in vitro data of Oscarson et al (1997), Thr-107 was found to be a substrate contacting residue in 11 of 13 models of the CYP2D6 active site developed using homology modeling and NMR studies of substrate binding (Modi et al, 1996).…”

“…Data from structural models and sequence alignment data indicate that Thr-107 and Arg-296 lie within distinct substrate contacting regions of the CYP2D6 active site (Hasemann et al, 1995;Modi et al, 1996). Consistent with the in vitro data of Oscarson et al (1997), Thr-107 was found to be a substrate contacting residue in 11 of 13 models of the CYP2D6 active site developed using homology modeling and NMR studies of substrate binding (Modi et al, 1996). Nevertheless, available population phenotyping studies and in vitro data suggest that the biotransformation of CYP2D6 substrates may be differentially affected by allelic variants present in individuals of African origin.…”

Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone

“…T 1 relaxation studies have been used successfully to estimate distances for codeine in CYP2D6 [20], diclofenac in CYP2C9 [19], caffeine in CYP1A2 [16], flurbiprofen and dapsone in CYP2C9 [12] and acetaminophen and caffeine in CYP3A4 [18]. The present study demonstrated that in all CYP2C9 proteins studied (CYP2C9.1, CYP2C9.2, CYP2C9.3 and CYP2C9.5) the flurbiprofen proton to heme iron distances, notably at the 4′-H which is the primary site of metabolism, though variable, did not directly correlate with previously observed differences in enzyme activity and correspondingly not directly with the amino acid substitutions in the variant proteins, either.…”

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

“…Purification was performed with nickel-chelate chromatography, essentially as described for human CYP2D6 [24] but with the modification that a mixture of emulgen 913 (2%) and Na-cholate (0.2%) was used for solubilization. CYP1A1 was electrophoretically homogeneous and had a specific P450 content of 11 nmolAEmg )1 protein.…”