Active-Site Topologies of Human CYP2D6 and Its Aspartate-301 → Glutamate, Asparagine, and Glycine Mutants

doi:10.1006/abbi.1996.0291

Archives of Biochemistry and Biophysics

Volume 331, Issue 1, 1 July 1996, Pages 134-140

https://doi.org/10.1006/abbi.1996.0291 Get rights and content

Abstract

Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5–7 Å from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, andp-biphenyldiazene, which react with P450 enzymes to form σ-bonded aryl–iron (Fe–Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces theN-arylprotoporphyrin IX regioisomers (N_B:N_A:N_C:N_D, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; andp-biphenyl, 16:84:00:00. These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in theN-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity. These results provide a refined model of the active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site.

References (0)

Cited by (44)

Topological role of cytochrome P450 2D6 active site residues
2006, Archives of Biochemistry and Biophysics
Citation Excerpt :
Membranes were resuspended in TSE buffer and stored at −80 °C until use. Formation of N-aryl-PPIX isomers was carried out by a procedure analogous to that of Mackman et al. [8]. Typically, 2–3 nmol of wild-type CYP2D6 or its I106E, F120A, D301Q, T309V, and T312V mutants was resuspended in 1 ml of potassium phosphate buffer (100 mM, pH 7.4).
Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.
Progress in cytochrome P450 active site modeling
2005, Archives of Biochemistry and Biophysics
Citation Excerpt :
A characteristic common to the vast majority of CYP2D6 substrates is the presence of at least one basic nitrogen atom. Many models of the active site of CYP2D6 have postulated the involvement of a carboxylate group in the protein forming a salt bridge with this basic nitrogen [51–55]—this has been proposed to be Asp301, a residue in the I-helix (substrate recognition site SRS4), both by modeling [53,56] and by mutagenesis [57]. Additionally, our models [56,58] were used in conjunction with sequence analysis to design a CYP2D6 mutant (F483I—based on an isoleucine in this position in testosterone-metabolizing CYP2D9) with novel specificity, able to metabolize testosterone [59].
Models capable of predicting the possible involvement of cytochromes P450 in the metabolism of drugs or drug candidates are important tools in drug discovery and development. Ideally, functional information would be obtained from crystal structures of all the cytochromes P450 of interest. Initially, only crystal structures of distantly related bacterial cytochromes P450 were available—comparative modeling techniques were used to bridge the gap and produce structural models of human cytochromes P450, and thereby obtain some useful functional information. A significant step forward in the reliability of these models came four years ago with the first crystal structure of a mammalian cytochrome P450, rabbit CYP2C5, followed by the structures of two human enzymes, CYP2C8 and CYP2C9, and a second rabbit enzyme, CYP2B4. The evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism, is presented as a case study.
Comparative molecular field analysis and QSAR on substrates binding to cytochrome P450 2D6
2003, Bioorganic and Medicinal Chemistry
In this study, we utilized comparative molecular field analysis (CoMFA) to gain a better understanding of the steric and electrostatic features of the cytochrome P450 2D6 (CYP2D6) active site. The training set consists of 24 substrates with reported K_M values from liver microsomal CYP2D6 spanning an activity range of almost three log units. The low energy conformers were fit by root mean square (RMS) to minaprine at the site of metabolism and to the protonated nitrogen. In this manner, we constructed two CoMFA models, one model with a distance constraint and another without. The model with the distance parameter (non-crossvalidated R²=0.99) was approximately equal to the CoMFA without a distance parameter (non-cross-validated R²=0.98). Validation of our CoMFA was accomplished by predicting the K_M values of 15 diverse CYP2D6 substrates not in the original training set resulting in a predictive R²=0.62. Finally, we also pursued correlations of pK_a and log P with CYP2D6 substrate K_M in an effort to investigate other physicochemical properties.
Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6
2003, Journal of Biological Chemistry
Citation Excerpt :
However, it should be noted that the K m value for diclofenac is significantly higher (36-fold) than the average reported values (33) and that the catalytic efficiency (k cat/K m) much lower (around 1%) than that of CYP2C9. CYP2D6 shows a clear, though not exclusive, preference for the metabolism of basic substrates that contain a nitrogen atom protonated at physiological pH. A common hypothesis has been that this may be due to electrostatic interactions of the protonated atom with the carboxylate group of Asp-301 (4, 16–20). Several studies have pointed to a possible role for a second carboxylate group, that of Glu-216, as a binding determinant, principally to explain the metabolism of the larger substrates with a basic nitrogen atom ≥10 Å from the site of oxidation (34–37).
Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216–231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K _m values increased 10–100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylationversus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50–75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K _m 108 μm,k _cat 5 min⁻¹) along with nifedipine (K _m 28 μm,k _cat 2 min⁻¹⁾ and tolbutamide (K _m 315 μm,k _cat 1 min⁻¹), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.
Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes
2003, Archives of Biochemistry and Biophysics
Citation Excerpt :
In this case, two phenylalanine residues are involved in making π–π contacts with the substrate and there is an ionic interaction between the positively charged protonated nitrogen atom of propranolol and an aspartate residue (Asp301) in the I helix. One of the phenylalanines [68] has been the subject of site-directed mutagenesis in CYP2D6, and the aspartate-301 residue has been extensively studied using this technique, clearly demonstrating its involvement in substrate binding [14,51]. Consequently, the CYP2D6 active site interaction exhibits a consistency with available experimental evidence from substrate metabolism and mutagenesis studies.
A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.
Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein
2001, Archives of Biochemistry and Biophysics
Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60–100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min⁻¹, respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The K_m and k_cat values for bufuralol metabolism were estimated to be 10.2 μM and 4.1 min⁻¹, respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2′-5′ ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min⁻¹, respectively. Bufuralol 1′-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6β-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts.

View all citing articles on Scopus

^☆: This work was supported by National Institutes of Health Grant GM25515 (P.R.O.M.) and the Wellcome Trust Grant 038735 (M.S.L., G.T.T., C.R.W.). Support for the Liver Center core facilities at the University of California, San Francisco, was provided by Grant 5 P30 DK26743.

²: To whom correspondence should be addressed at School of Pharmacy, University of California, San Francisco, CA 94143-0446.

View full text

Regular ArticleActive-Site Topologies of Human CYP2D6 and Its Aspartate-301 → Glutamate, Asparagine, and Glycine Mutants☆

Abstract

Regular Article
Active-Site Topologies of Human CYP2D6 and Its Aspartate-301 → Glutamate, Asparagine, and Glycine Mutants☆