cytochrome p450 superfamily in Arabidopsis thaliana: isolation of cDNAs,Differential Expression,and RFLP mapping of multiple cytochromes P450

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.     

Crystal structures of cytochrome P450nor and its mutants (Ser286-->Val, Thr) in the ferric resting state at cryogenic temperature: a comparative analysis with monooxygenase cytochrome P450s

Cytochrome P450nor (P450nor) is a heme enzyme isolated from the denitrifying fungus Fusarium oxysporum and catalyzes the NO reduction to N2O. Crystal structures of the wild type and two Ser286 mutants (Ser286-->Val, Ser286-->Thr) of P450nor have been determined for the ferric resting forms at a 1.7 A resolution at cryogenic temperature (100 K). We carried out three comparative analyses: (1) between the structures of P450nor at room temperature and cryogenic temperature, (2) between the structures of P450nor and four monooxygenase P450s, and (3) between the structures of the WT and the Ser286 mutant enzymes of P450nor. Comparison of the charge distribution on the protein surface suggests that proton and electron flow to the heme site is quite different in P450nor than in monooxygenase P450s. On the basis of the mutant structures, it was found that a special hydrogen-bonding network, Wat99-Ser286-Wat39-Asp393-solvent, acts as a proton delivery pathway in NO reduction by P450nor. In addition, the positively charged cluster located beneath the B'-helix is suggested as possible NADH binding site in P450nor, from which the direct two-electron transfer to the heme site allows to generate the characteristic intermediate in the NO reduction. These structural characteristics were not observed in structures of monooxygenase P450s, implying that these are factors determining the unique NO reduction activity of P450nor.     

Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features

Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.     

Cytochrome P450

Since 1993, three new cytochrome P450 X-ray structures have been determined, giving a total of four known structures. Two of the new structures are in the substrate-free form and one is substrate-bound. These new structures, together with a wealth of mutagenesis studies on various P450s, have provided considerable information on what structural features control substrate specificity in P450s. In addition, some important insights into the catalytic mechanism have been made.     

Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel

Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analogue tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations: closed (P450cam-C), intermediate (P450cam-I), and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of helix F, while both helices F and G move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well-defined states, while P450cam-O shows evidence of a somewhat broader distribution of conformations and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multistep closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well-defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multistep pathway may have significant implications for a full understanding of substrate specificity, kinetics, and coupling of substrate binding to P450 function.     

Crystal structures of epothilone D-bound,epothilone B-bound,and substrate-free forms of cytochrome P450epoK

Epothilones are potential anticancer drugs that stabilize microtubules by binding to tubulin in a manner similar to paclitaxel. Cytochrome P450epoK (P450epoK), a heme containing monooxygenase involved in epothilone biosynthesis in the myxobacterium Sorangium cellulosum, catalyzes the epoxidation of epothilones C and D into epothilones A and B, respectively. The 2.10-, 1.93-, and 2.65-A crystal structures reported here for the epothilone D-bound, epothilone B-bound, and substrate-free forms, respectively, are the first crystal structures of an epothilone-binding protein. Although the substrate for P450epoK is the largest of a P450 whose x-ray structure is known, the structural changes along with substrate binding or product release are very minor and the overall fold is similar to other P450s. The epothilones are positioned with the macrolide ring roughly perpendicular to the heme plane and I helix, and the thiazole moiety provides key interactions that very likely are critical in determining substrate specificity. Interestingly, there are strong parallels between the epothilone/P450epoK and paclitaxel/tubulin interactions. Based on structural similarities, a plausible epothilone tubulin-binding mode is proposed.     

Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at "hot spots" of protein-protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.     

The ins and outs of cytochrome P450s

The active site of cytochromes P450 is situated deep inside the protein next to the heme cofactor. Consequently, enzyme specificity and kinetics can be influenced by how substrates pass through the protein to access the active site and how products egress from the active site. We previously analysed the channels between the active site and the protein surface in P450 crystal structures available in October 2003 [R.C. Wade, P.J. Winn, I. Schlichting, Sudarko, A survey of active site access channels in cytochromes P450, J. Inorg. Biochem. 98 (2004) 1175-1182]. Since then, 52 new P450 structures have been made available, including entries for ten isozymes for which structures were not previously available. We present an updated survey covering all P450 crystal structures available in March 2006. This survey shows channels not observed earlier in crystal structures, some of which were identified in previous molecular dynamics simulations. The crystal structures demonstrate how some of the channels can merge when the protein structure opens up resulting in a wide cleft to the active site, caused largely by movements of the F-G helix-loop-helix and the B-C loop. Significant differences were observed between the channels in the crystal structures of the mammalian and bacterial enzymes. The multiplicity of channels suggests possibilities for substrate channelling to and from the P450s.     

Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects. Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length. Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site. However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure. The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe. Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage. The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures. The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site. They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s. Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures.     

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.     

Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants

Terpenoids are an extensive and diverse group of plant secondary metabolites. To date, they have been applied in many fields including industry, medicine and health. The wide variety of terpenoid compounds cannot arise solely from simple cyclisations of a precursor molecule or from a single-step reaction; their structural diversity depends on the modification of many specific chemical groups, rearrangements of their skeletal structures and on the post-modification reactions. Most of the post-modification enzymes that catalyse these reactions are cytochrome P450 monooxygenases. Therefore, the discovery and identification of plant P450 genes plays a vital role in the exploration of terpenoid biosynthesis pathways. This review summarises recent research progress relating to the function of plant cytochrome P450 enzymes, describes P450 genes that have been cloned from full-length cDNA and identifies the function of P450 enzymes in the terpenoid biosynthesis pathways of several medicinal plants.     

Automated multiple analysis of protein structures: Application to homology modeling of cytochromes P450

A computational strategy for homology modeling, using several protein structures comparison, is described. This strategy implies a formalized definition of structural blocks common to several protein structures, a new program to compare these structures simultaneously, and the use of consensus matrices to improve sequence alignment between the structurally known and target proteins. Applying this method to cytochromes P450 led to the definition of 15 substructures common to P450_cam, P450_BM3, and P450_terp, and to proposing a 3D model of P450_eryF. Proteins 28:388–404, 1997 © 1997 Wiley-Liss, Inc.     

A three-dimensional model of aromatase cytochrome P450.

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.     

A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.     

Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.     

FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.     

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme.