Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.     

Effect of carbonate anion on cytochrome P450 2D6-mediated metabolism in vitro: the potential role of multiple oxygenating species

Studies were designed to investigate various anions and their effects on cytochrome P450 2D6-mediated metabolism in vitro. Incubations were initially performed in buffered phosphate, carbonate, sulfate, and acetate solutions (50mM, pH 7.4), with CYP2D6 substrates dextromethorphan, 7-methoxy-4-(aminomethyl)-coumarin (MAMC), (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride [(-)-OSU6162], and amitriptyline. Dextromethorphan and MAMC O-dealkylation activity in buffered carbonate was approximately 25 and 38%, respectively, relative to phosphate, while activity in sulfate and acetate buffers displayed minor differences. In contrast, N-dealkylation reactions for both (-)-OSU6162 and amitriptyline were unaffected by the presence of carbonate, and the other anions tested. Subsequent kinetic studies revealed that the basis of reduced turnover of dextromethorphan was primarily a V(max) effect, as the V(max) for the rate was 16.9 and 5.6 pmol/min/pmol P450 in phosphate and carbonate, respectively. Interestingly, similar rates of dextromethorphan O-demethylation in phosphate and carbonate were observed when reactions were supported by cumene hydroperoxide (CuOOH). Furthermore, it was observed that while CuOOH could equally support dextromethorphan O-demethylation compared to NADPH, amitriptyline N-demethylation was only minimally supported. Finally, intramolecular kinetic isotope effect (KIE) experiments with amitriptyline-d3 in CuOOH-supported reactions yielded a k(H)/k(D) of 5.2, substantially higher than in phosphate and carbonate supported by NADPH (k(H)/k(D)=1.5). Overall, results suggest that carbonate disrupts the relative ratios of the potential P450 oxygenating species, which differentially catalyze O- and N-dealkylation reactions mediated by CYP2D6.     

Genetic variants in the cytochrome P450 2D6 gene in the Sri Lankan population

INTRODUCTION:

Cytochrome P450 2D6 (CYP2D6) enzymes are involved in the metabolism of a large number of commonly prescribed drugs such as antidepressants and cardiovascular drugs. The CYP2D6 *3, *4 and *14 variants associated with the loss of enzyme function; CYP2D6 *10 and *17 variants with reduced enzyme function; and CYP2D6 *2 variant with no effect on enzyme function. Establishing the frequency of these variant alleles in Sri Lankan population would be useful for optimizing pharmacotherapy with CYP2D6-substrate drugs.

OBJECTIVE:

The objective of this study was to determine the prevalence of CYP2D6 *2, *3, *4, *10, *14 and *17 variants in the main ethnic groups in the Sri Lankan population.

MATERIALS AND METHODS:

A total of 90 deoxyribonucleic acid (DNA) samples (30 each from Sinhalese, Tamils and Moors) were selected from a DNA resource at the Human Genetic Unit, Faculty of Medicine, University of Colombo. This collection had been made for population genetic studies from a random population based volunteers. Genotyping was performed using published polymerase chain reaction/restriction fragment length polymorphism methods.

RESULTS:

The prevalence of the CYP2D6 variants in Sinhalese, Sri Lankan Tamils and Moors respectively were CYP2D6 *2: 37%, 41.6% and 37.9%; CYP2D6 *3: 60.3%, 45% and 30%; CYP2D6 *4: 21.6%, 6.6% and 8.3%; CYP2D6 *10: 40%, 35% and 44%. CYP2D6 *14 and *17 variants were not identified.

CONCLUSION:

CYP2D6*3, *4 and *10 variants, which are associated with reduced or loss of CYP2D6 enzyme function were found in our population in significant frequencies. CYP2D6*4, which is reported to be a Caucasian variant was also found in all three ethnic groups.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.     

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.     

细胞色素P450 2D6酶缺陷等位基因的分析

细胞色素P450 2D6(CYP2D6)第1 795位胸腺嘧啶核苷缺失造成CYP2D6酶活性缺陷,该等位基因被称为CYP2D6T.对该等位基因的测定有助于准确预测CYP2D6表现型.利用等位基因特异扩增法的基本原理,建立了测定CYP2D6T的方法.经396例测定,证明比利用PCR扩增后再酶切的方法更为快捷、更少污染,为该项测定应用于临床奠定基础.     

细胞色素P450 2D6缺陷型等位基因的家系分析

利用等位基因特民扩增法(ASA)为基础的基因分型法,对细胞色素P4502D6 (CYP2D6)缺陷型等位基因携带者的9个家庭共38个进行了基因分型,并与用右旋美沙芬为 探针的表型分型法进行对比,发现两种方法的结果是一致的,CYP2D6酶缺陷型等位基因呈常染色体隐性遗传。 Abstract:A genotyping method based on the principle of allele-specific amplification and a phenotyping procedure with dextromethorphan as a probe were employed in familial study of nine families with 38 members for the cytochrome P450 2D6(CYP2D6)deficient alleles——CYP2D6A,CYP2D6B,CYP2D6D and CYP2D6T.The results showed that the CYP2D6 deficient alleles were inherited as an autosomal recessive trait.     

Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for the first time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe120 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe120 in binding dextromethorphan and MDMA.     

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Alois Bonifacio, André R. Groenhof and Peter H. J. Keizers contributed equally to this work.     

Role of the conserved threonine 309 in mechanism of oxidation by cytochrome P450 2D6

Based on sequence alignments and homology modeling, threonine 309 in cytochrome P450 2D6 (CYP2D6) is proposed to be the conserved I-helix threonine, which is supposed to be involved in dioxygen activation by CYPs. The T309V mutant of CYP2D6 displayed a strong shift from O-dealkylation to N-dealkylation reactions in oxidation of dextromethorphan and 3,4-methylenedioxymethylamphetamine. This may be explained by an elevated ratio of hydroperoxo-iron to oxenoid-iron of the oxygenating species. In consistence, using cumene hydroperoxide, which directly forms the oxenoid-iron, the T309V mutant again selectively catalyzed the O-dealkylation reactions. The changed ratio of oxygenating species can also explain the decreased activity and changed regioselectivity that were observed in 7-methoxy-4-(aminomethyl)-coumarin and bufuralol oxidation, respectively, by the T309V mutant. Interestingly, the T309V mutant always showed a significantly increased, up to 75-fold, higher activity compared to that of the wild-type when using cumene hydroperoxide. These results indicate that T309 in CYP2D6 is involved in maintaining the balance of multiple oxygenating species and thus influences substrate and regioselectivity.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

Metabolons involving plant cytochrome P450s

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.     

Cooperativity of cytochrome P450 1A2: interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)₂) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262-7272]. Multiphasic kinetics were observed for the binding of Ph(NC)₂ to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)₂ induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)₂ binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)₂ was bound. No oxidation products of Ph(NC)₂ were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2·α-naphthoflavone (αNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)₂; in the case of αNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)₂ inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.     

Molecular modeling and identification of substrate binding site of orphan human cytochrome P450 4F22

Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human cytochrome P450 4F22 (CYP4F22) was categorized into "orphan" CYPs because of its unknown function. CYP4F22 is a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22 remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the biological roles of CYP4F22 and structure-based drug design.     

Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HSPs are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.     

细胞色素P450超家族蛋白质基于结构知识的序列联配

应用计算机程序对细胞色素Ｐ４５０蛋白２０个家族的代表序列进行初步多序列联配后,利用结构知识对匹配结果加以调整,调整的依据是Ｐ４５０蛋白二级结构特征和某些超二级结构特征的保守性,三维结构空间配置的保守性,在结构与功能重要残基的保守性,亲水和疏水片段的保守性。大量定点突变实验、选择性化学修饰实验、抗多肽抗体结合实验等能很好地确证该联配结果的正确性。联配结果对真核Ｐ４５０蛋白膜结合方式、活性位点残基分布、与氧化还原偶联蛋白有相互作用的残基的分布等作出了合理的推断。细胞色素Ｐ４５０超家族蛋白基于结构知识的序列联配的尝试对Ｐ４５０蛋白结构与功能关系研究提供了参考     

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.     

Progress in cytochrome P450 active site modeling

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.     

Measurement of immunoreactive cytochrome P450 2E1 in human leucocytes

We describe initial results on a Western blotting method, using a ployclonal antibody and chemiluminescence detection, for the measurement of cytochrome P450 2E1 in human lymphocytes. The method has been used to study the levels of 2E1 in lymphocytes isolated from 5 ml blood samples collected from a small group of well-controlled type 1 diabetics and healthy individuals. The described method offers increased sensitivity compared with a previously published method and does not need in vitro culturing of the lymphocytes prior to 2E1 measurement. The apparent molecular weight of the lymphocyte P450 2E1 was 55 kDa. There was approximately a six-fold difference in expression levels of 2E1 detected by this immunochemical technique across the study population.