Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site

A 2.7-Angstrom molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the beta1 sheet and helix F' that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F', G', and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism.     

Tight-binding inhibition by alpha-naphthoflavone of human cytochrome P450 1A2

Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that alpha-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (K(i)=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (K(i)=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate.     

Determinants of cytochrome P450 2C8 substrate binding: structures of complexes with montelukast, troglitazone, felodipine, and 9-cis-retinoic acid

Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 A), troglitazone (2.7 A), felodipine (2.3 A), and 9-cis-retinoic acid (2.6 A) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.     

Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.     

The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 A by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.     

Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes

The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 A resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 A from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 A from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a approximately 1.5 A movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F' and G'. The G' helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes.     

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

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.     

Structural Characterization of Human Cytochrome P450 2C19: ACTIVE SITE DIFFERENCES BETWEEN P450s 2C8, 2C9, AND 2C19*?

To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme.     

Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding

The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM and k(cat) values of 13 and 16 min(-1), respectively. Spectrally determined binding constants are similar to the K(m) values. The structure indicates that the pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.     

Differences in flexibility of active sites of cytochromes P450 probed by resonance Raman and UV-Vis absorption spectroscopy.

Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.     

Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.     

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.     

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides

BackgroundCytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure.MethodsThe metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures.ResultsLower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F–G loop regions and active site cavity volumes for the F87A mutated structures.ConclusionsAnalysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site.General significanceThe metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.     

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.     

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.     

Unusual regioselectivity and active site topology of human cytochrome P450 2J2

The oxidation of six derivatives of terfenadone by recombinant human CYP2J2 (CYP = cytochrome P450) was studied by high-performance liquid chromatography coupled to mass spectrometry (MS) using tandem MS techniques and by 1H NMR spectroscopy. CYP2J2 exhibited a surprising regioselectivity in favor of the hydroxylation of the substrate terminal chain at the weakly reactive homobenzylic position. In contrast, hydroxylation of the same substrates by CYP3A4 mainly occurred on the most chemically reactive sites of the substrates (N-oxidation and benzylic hydroxylation). A 3D homology model of CYP2J2 was constructed using recently published structures of CYP2A6, CYP2B4, CYP2C8, CYP2C9, and CYP2D6 as templates. In contrast with other CYP2 structures, it revealed an active site cavity with a severely restricted access of substrates to the heme through a narrow hydrophobic channel. Dynamic docking of terfenadone derivatives in the CYP2J2 active site allowed one to interpret the unexpected regioselectivity of the hydroxylation of these substrates by CYP2J2, which is mainly based on this restricted access to the iron. The structural features that have been found to be important for recognition of substrates or inhibitors by CYP2J2 were also interpreted on the basis of CYP2J2-substrate interactions in this model.     

Pivotal role of water in the mechanism of P450BM-3

Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 A of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.     

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.     

细胞色素P450 2B4的结构及其催化反应

细胞色素P450是广泛存在于动物、植物和微生物中的含亚铁血红素单加氧酶,参与致癌作用和药物代谢、类固醇激素合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子等生理过程。P450能够催化完成伯、仲碳氢键羟基化、烯烃和芳烃环氧化、碳碳键耦合和断裂、α羟基化(去烷基化和杂原子氧化)、还原、1,2-迁移(卤素、氢和苯)等有机反应。本文综述了P450 2B4的结构与功能,讨论了细胞色素P450 2B4的活性中心和底物识别位点、与底物反应和产物释放的机理,以及P450在有机合成中的应用。