Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104

CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single specific primer PCR was applied to yield the whole gene sequence via chromosomal walks. CYP153 consists of 497 amino acids (M(r) = 56 kDa) and thus represents an unusually long bacterial P450, containing all P450 typical structural elements. It constitutes the new P450 family CYP153. The prolonged N-terminus of about 90 amino acids does not contain a so far known membrane-anchoring sequence but a 28-amino acid long amphipathic helix. The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loop is discussed with respect to substrate binding and recognition.     

Crystal structure of the human prostacyclin synthase

Prostacyclin synthase (PGIS) catalyzes an isomerization of prostaglandin H(2) to prostacyclin, a potent mediator of vasodilation and anti-platelet aggregation. Here, we report the crystal structure of human PGIS at 2.15 A resolution, which represents the first three-dimensional structure of a class III cytochrome P450. While notable sequence divergence has been recognized between PGIS and other P450s, PGIS exhibits the typical triangular prism-shaped P450 fold with only moderate structural differences. The conserved acid-alcohol pair in the I helix of P450s is replaced by residues G286 and N287 in PGIS, but the distinctive disruption of the I helix and the presence of a nearby water channel remain conserved. The side-chain of N287 appears to be positioned to facilitate the endoperoxide bond cleavage, suggesting a functional conservation of this residue in O-O bond cleavage. A combination of bent I helix and tilted B' helix creates a channel extending from the heme distal pocket, which seemingly allows binding of various ligands; however, residue W282, placed in this channel at a distance of 8.4 A from the iron with its indole side-chain lying parallel with the porphyrin plane, may serve as a threshold to exclude most ligands from binding. Additionally, a long "meander" region protruding from the protein surface may impede electron transfer. Although the primary sequence of the PGIS cysteine ligand loop diverges significantly from the consensus, conserved tertiary structure and hydrogen bonding pattern are observed for this region. The substrate-binding model was constructed and the structural basis for prostacyclin biosynthesis is discussed.     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.     

Structural characterization of the complex between alpha-naphthoflavone and human cytochrome P450 1B1

The atomic structure of human P450 1B1 was determined by x-ray crystallography to 2.7 Å resolution with α-naphthoflavone (ANF) bound in the active site cavity. Although the amino acid sequences of human P450s 1B1 and 1A2 have diverged significantly, both enzymes exhibit narrow active site cavities, which underlie similarities in their substrate profiles. Helix I residues adopt a relatively flat conformation in both enzymes, and a characteristic distortion of helix F places Phe²³¹ in 1B1 and Phe²²⁶ in 1A2 in similar positions for π-π stacking with ANF. ANF binds in a distinctly different orientation in P450 1B1 from that observed for 1A2. This reflects, in part, divergent conformations of the helix B′-C loop that are stabilized by different hydrogen-bonding interactions in the two enzymes. Additionally, differences between the two enzymes for other amino acids that line the edges of the cavity contribute to distinct orientations of ANF in the two active sites. Thus, the narrow cavity is conserved in both P450 subfamily 1A and P450 subfamily 1B with sequence divergence around the edges of the cavity that modify substrate and inhibitor binding. The conservation of these P450 1B1 active site amino acid residues across vertebrate species suggests that these structural features are conserved.     

Molecular cloning and expression of CYP6B8: a xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea

Xanthotoxin, a plant allelochemical, induces alpha-cypermethrin insecticide tolerance in Helicoverpa zea (corn earworm); inhibition of tolerance by piperonyl butoxide implicates cytochrome P450 monooxygenases (P450s) in the detoxification of this insecticide. To characterize the xanthotoxin-inducible P450 that might mediate alpha-cypermethrin tolerance in this species, a cDNA library prepared from xanthotoxin-induced H. zea fifth instar larvae was screened with cDNAs encoding furanocoumarin-metabolizing P450s from Papilio polyxenes (CYP6B1v2) and P. glaucus (CYP6B4v2) as well as a sequence-related P450 from Helicoverpa armigera (CYP6B2). One full-length cDNA isolated in this screening shares 51-99% amino acid identity with the CYP6B subfamily of P450s isolated from Papilio and Helicoverpa species and, thus, has been designated CYP6B8. All of these CYP6B subfamily members share a number of highly conserved domains, including substrate recognition site 1 (SRS 1) that is critical for xanthotoxin metabolism by CYP6B1v2 from Papilio polyxenes and coumarin metabolism by CYP2a5 from Mus musculus. Northern and RT-PCR analyses indicate that CYP6B8 expression is strongly induced by xanthotoxin and phenobarbital and negligibly induced by alpha-cypermethrin.     

Preliminary characterization and crystal structure of a thermostable cytochrome P450 from Thermus thermophilus

The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-A resolution. The overall P450 structure remains conserved despite the low sequence identity between the various P450s. The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability. The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single charge-charge interactions. Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.     

Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus

The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.     

Crystal structures of substrate-free and retinoic acid-bound cyanobacterial cytochrome P450 CYP120A1

The crystal structures of substrate-free and all-trans-retinoic acid-bound CYP120A1 from Synechocystis sp. PCC 6803 were determined at 2.4 and 2.1 A resolution, respectively, representing the first structural characterization of a cyanobacterial P450. Features of CYP120A1 not observed in other P450 structures include an aromatic ladder flanking the channel leading to the active site and a triple-glycine motif within SRS5. Using spectroscopic methods, CYP120A1 is shown to bind 13-cis-retinoic acid, 9-cis-retinoic acid, and retinal with high affinity and dissociation constants of less than 1 microM. Metabolism of retinoic acid by CYP120A1 suggests that CYP120A1 hydroxylates a variety of retinoid derivatives in vivo. On the basis of the retinoic acid-bound CYP120A1 crystal structure, we propose that either carbon 2 or the methyl groups (C16 or C17) of the beta-ionone ring are modified by CYP120A1.     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

Molecular dynamics analysis reveals structural insights into mechanism of nicotine N-demethylation catalyzed by tobacco cytochrome P450 mono-oxygenase

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys-trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.     

Structures of human cytochrome P-450 2E1. Insights into the binding of inhibitors and both small molecular weight and fatty acid substrates

Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.     

Sterol 14alpha-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.     

Conservation in the CYP51 family. Role of the B' helix/BC loop and helices F and G in enzymatic function

CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.     

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.     

Folding requirements are different between sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis and human or fungal orthologs

Upon sequence alignment of CYP51 sterol 14alpha-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting beta-strand 3-2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

The 1.92-A structure of Streptomyces coelicolor A3(2) CYP154C1. A new monooxygenase that functionalizes macrolide ring systems

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O(2)-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code ). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional beta-strand transforming the five-stranded beta-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactones in vitro.