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1.
Cytochrome P450 总被引：1，自引：0，他引：1
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. 相似文献
2.
Feldmann KA 《Current opinion in plant biology》2001,4(2):162-167
In the past year, several cytochrome P450 genes have been identified that will be important for generating crop protectants and natural medicinal products. Among these are the 2-hydroxyisoflavone synthase (CYP93C) and the indole-3-acetaldoxime N-hydroxylase (CYP83B1) genes, which catalyze the formation of isoflavones and glucosinolates, respectively. 相似文献
3.
植物细胞色素P450 总被引：11，自引：0，他引：11
对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。 相似文献
4.
David C. Lamb Li Lei Andrew G. S. Warrilow Galina I. Lepesheva Jonathan G. L. Mullins Michael R. Waterman Steven L. Kelly 《Journal of virology》2009,83(16):8266-8269
The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes (YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.Cytochromes P450 (CYP) are a superfamily of heme-thiolate enzymes that are distributed widely throughout Eukarya, Archaea, and Bacteria (http://drnelson.utmem.edu/CytochromeP450.html). Viruses are the most abundant biological entities in nature and are also responsible for many diseases in plants and animals. To date, 2,180 viral genome sequencing projects have been completed and annotated and no CYP open reading frames have been observed (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10239&opt=Virus).Acanthamoeba polyphaga mimivirus is the largest known virus, which grows in amoeba (5). In 2004, the 1.2-Mbp genome of mimivirus (GenBank accession no. AY653733) was sequenced (9). Its genome is larger than that of several bacteria and archaea and is predicted to encode 911 proteins, among which only 298 have predicted functions. Many atypical proteins are predicted to be encoded by the mimivirus genome, including key protein translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (9). Interestingly, among genes never yet reported to occur in a virus, mimivirus contained two putative gene sequences predicted to encode cytochrome P450 enzymes (GenBank accession no. YP_142886 and YP_143162, also known as MIMI_L532 and MIMI_L808, respectively). First, YP_142886 is a putative protein of 468 amino acids in length. In a BLASTP search, this putative CYP protein showed homology to a range of bacterial P450 proteins, including a P450 protein from Chloroflexus aurantiacus (23% identity) and CYP171 from Streptomyces peucetius (23% identity). Additionally, YP_142886 also showed homology at the same level to nematode P450 proteins, including Caenorhabditis briggsae CYP37B1 (25% identity) and a P450 protein from the sea squirt Ciona intestinalis similar to the CYP4 family (24% identity). Efforts in our laboratory to express the YP_142886 gene and verify that it indeed encodes a cytochrome P450 have been unsuccessful, but additional attempts are in progress. The mimivirus protein YP_143886 was designated CYP5254A1 by David Nelson (http://drnelson.utmem.edu/CytochromeP450.html).The second putative mimivirus CYP protein (YP_143162) showed in a BLASTP search the strongest homology to CYP51 proteins (7) from a variety of organisms, including protozoal CYP51 proteins from, e.g., Leishmania major (23% identity); plant CYP51 proteins from, e.g., Arabidopsis thaliana (22% identity); and fungal CYP51 proteins from, e.g., Aspergillus fumigatus (21% identity). This homology is low, strongly suggesting the absence of a functional link. Further analysis of the YP_143162 709-amino-acid sequence revealed this putative CYP protein to be approximately 200 residues longer than its closest CYP homologues, and this protein was proposed to comprise a fused protein domain of unknown function, with the best homologies to lipopolysaccharide core biosynthesis glycosyl transferase from Proteus mirabilis HI4320 (26% identity), integral membrane sensor signal transduction histidine kinase from Dinoroseobacter shibae DFL 12 (24% identity), a short region of dysferlin from Strongylocentrotus purpuratus (35% identity), and pierisin-1 (NAD-DNA ADP-ribosyltransferase) from Pieris rapae (24% identity). Interestingly, several putative posttranslational modifications, including one N glycosylation site, a protein kinase C phosphorylation site, four casein kinase II phosphorylation sites, and three myristoylation sites, were predicted to exist in this C-terminal extension peptide, representing the first time these specific modifications were present in a P450 molecule.Historically, a protein can be identified as a cytochrome P450 through the production of the carbon monoxide (CO)-bound form of the reduced (sodium dithionite-treated) pigment, which has an intense absorption band at 450 nm (8). Following isopropyl β-d-thiogalactopyranoside-induced expression of the full-length YP_143162 gene, utilizing the T7 promoter of the Escherichia coli expression vector pET17b (Novagen), only a protein producing a Soret maximum at 420 nm, recognized as the misfolded, incorrect form of CYP, was detected in reduced-difference CO spectrophotometry. Alterations in temperature, coexpression with molecular chaperones GroES and GroEL of E. coli (which allow production of active and correctly folded human P450s [4]), and the use of different E. coli strains for recombinant protein expression did not produce correctly folded YP_143162 (data not shown). Analysis of the primary sequence revealed the presence of a putative membrane-spanning segment located from residue 2 to 19 which may interfere with the expression of correctly folded P450 (1). A modified YP_143162 gene sequence encoding an insertion of alanine at amino acid position 2 was generated by PCR. This N-terminally truncated enzyme (Fig. (Fig.1A),1A), expressed as a correctly folded CYP protein, generated a characteristic reduced-CO-difference spectrum with a maximum at 448 nm (Fig. (Fig.1B).1B). Cell fractionation revealed the truncated protein to be associated with the membrane fraction following ultracentrifugation at 100,000 × g, and no CYP protein was detected in the E. coli cytoplasm, thus necessitating the use of detergents to purify the enzyme. The truncated but membrane-bound enzyme was expressed at CYP levels of >1,000 nmol P450/liter of culture, with supplementation of the growth medium with the heme precursor δ-aminolevulinic acid increasing heme-incorporated CYP expression levels approximately twofold, to 2,000 to 3,000 nmol P450/liter of culture. Truncated YP_143162 was the major band observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Fig. (Fig.1C),1C), with the molecular mass estimated to be 78 kDa, in agreement with the predicted molecular mass of the truncated protein. The absolute absorbance spectrum of the purified (oxidized, Fe3+) YP_143162 protein showed a Soret band at 419 nm and α and β bands at 572 and 536 nm, while reduction with sodium dithionite (Fe2+) resulted in a typical Soret peak shift to 417 nm. Most CYP enzymes are purified in a low-spin state with a water molecule hexacoordinated to the CYP heme iron, as indicated by a peak at 390 nm in the absolute spectrum. Substrate or inhibitor addition shifts the heme to the high-spin state, as indicated by a peak at 419 nm in the absolute spectrum, which can be the case when imidazole, used to purify the protein, binds to the heme iron. Continued dialysis for removal of the imidazole from the protein resulted in a shift from a high- to a low-spin state. Furthermore, quantification of the iron content (2) of YP_143162 (0.97 ± 0.06 atoms of iron per heme-containing molecule of YP_143162) indicated that there is one atom of iron per heme-containing molecule of YP_143162, confirming one atom of iron associated with the heme of this P450 protein. Given the weak homology of YP_143162 to sterol-metabolizing CYP proteins, the binding of the sterols lanosterol and obtusifoliol as well as the final A. polyphaga sterol end product ergosterol to purified enzyme was investigated as previously described (3). No evidence of sterol binding or metabolism was obtained (data not shown). Such data can be confirmed by the fact that the key motif aGQHTSs (which is involved in catalysis, includes an invariant H in all CYP51 proteins to date [6], and corresponds to a negatively charged residue [D/E] in other P450 families) is missing in YP_143162 (Fig. (Fig.2A).2A). Additionally, YP_143162 did not cluster with any CYP51 proteins but mapped to a distinct and separate branch on the tree (Fig. (Fig.2B).2B). A homology model was generated for mimivirus YP_143162 protein on the basis of the resolved P450 crystal structures of Mycobacterium tuberculosis CYP51 (Protein Data Bank accession no. 1E9X) and flavocytochrome CYP102A1 from Bacillus megaterium (Protein Data Bank accession no. 1JPZ). YP_143162 is predicted to adopt a typical P450 fold reflecting the similarity in helix assignment. It was possible to confirm the likely heme-coordinating residue (C425), and the EXXR motif, present in nearly all P450 proteins and involved in heme binding and P450 architecture, is also conserved in YP_143162 (Fig. (Fig.3).3). Consequently, the mimivirus protein YP_143162 was designated CYP5253A1 (http://drnelson.utmem.edu/CytochromeP450.html).Open in a separate windowFIG. 1.Purification and spectral characterization of mimivirus YP_143162. (A) N-terminal sequence of the full-length native enzyme, with the hydrophobic stretch that most likely forms a transmembrane α helix underlined. Below is shown the N-terminal sequence of the truncated CYP protein used to obtain the correctly folded P450 protein. (B) Absolute oxidized and reduced CO difference (inset) spectra at 1 μM P450 concentration. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10%). Lane 1, rainbow marker; lane 2, purified mimivirus CYP51-like protein.Open in a separate windowFIG. 2.Alignment of mimivirus YP_143162 with 143 CYP51 family members (only two representatives of each biological kingdom are shown). (A) The fragments shown are the BC loop (SRS1 region, helices F and G [SRS2 and SRS3] and helix I [SRS4]). (B) Representative phylogenetic tree of CYP51 sequences showing the position of YP_143162.Open in a separate windowFIG. 3.Mimivirus P450 model with marked secondary structural elements. The resultant homology models were validated by cross-reference to the secondary structure predictions. Homology models were generated with 10 iterations of the MODELLER program, and the model structure was clipped to the first 480 amino acids, for which the sequence identity with the resolved crystal structures of MTCYP51 and flavocytochrome P450-2 of Bacillus megaterium for the CYP domain was 16%. The energy-minimized model has very good ProsaII and Profiles 3D scores.The presence of genes encoding CYP in the mimivirus genome is intriguing from the standpoint of P450 evolutionary discussion. Phylogenetically, mimivirus and other giant viruses are very old and are thought to have existed prior to cellular organisms (9). Furthermore, it was previously proposed that a form of cytochrome P450 has been present in life forms for billions of years and before the advent of free atmospheric oxygen (10). Subsequently P450 had protective value in detoxifying reactive oxygen species and was retained in aerobic organisms as a monooxygenase in biosynthetic processes and in the degradation of complex molecules. Consequently, it can be speculated that P450 may have been present in a viral genome prior to the establishment of the three domains of life (eukaryotes and prokaryotes, which consist of bacteria and archaea). It is possible that mimivirus acquired CYP genes from a more ancient progenitor. Conversely, Moreira and Brochier-Armanet (7) hypothesize that the diverse mimivirus genes, many with eukaryotic homology, including CYP, were obtained by horizontal gene transfer. The basis of this theory suggests that the mimivirus host, A. polyphaga, is also host to parasitic, bacterial endosymbionts, including many Mycobacteria spp. which have high numbers of CYP genes within their genomes. Consequently, mimivirus may have picked up genes from such endosymbionts or from the amoeba host itself. At present, no Acanthamoeba genome has been sequenced to allow comparisons, but Acanthamoeba genomes certainly contain CYP51 for synthesis of sterols. Finally, although mimivirus was first isolated from A. polyphaga, additional hosts acting as a source of new genetic material for this virus cannot be ruled out. For example, mimivirus has been implicated as a causative agent of influenza in mice and humans, suggesting mammalian cellular hosts for mimivirus. It can also be argued that mimivirus CYP has evolved into a gene encoding a protein with a totally different function, unrelated to its being a P450. It is probable that we will never know the nature of the ancient P450 ancestor or how it evolved into the superfamily that we see today (>8,500 genes). However our confirmation of a viral gene encoding a cytochrome P450 protein invigorates this continuing debate. 相似文献
5.
Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice. 相似文献
6.
Fumiko Mitani 《Molecular and cellular biochemistry》1979,24(1):21-43
Summary Cytochrome P450 in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P450 each catalyzing a specified reaction have been purified to a homogeneous state, that is, P450scc having cholesterol side chain cleavage activity and P45011 having 11-hydroxylation activity. The properties of these purified P450's as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P450 in the mitochondria) preparation in situ.Among many findings, both purified cytochromes P450 were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P450. These effects are important from the point of view that the primary role of the heme of cytochrome P450 is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P450 in the reconstituted system have also been described Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method. 相似文献
7.
Mary A. Schuler 《植物科学评论》1996,15(3):235-284
Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated. 相似文献
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9.
Cytochrome P450s in flavonoid metabolism 总被引：2，自引：0，他引：2
In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed. 相似文献
10.
细胞色素P450与肿瘤 总被引：4，自引：0，他引：4
本文综棕了细胞色素Ｐ４５０同工酶与致癌物代谢、与抗癌药的相互作用以及化的关系,并对调控Ｐ４５０同工酶以防治肿瘤的策略进行了论述。由于Ｐ４５０同工酶具有多态性、工物特异性及可诱导性的特点,在调控Ｐ４５０同工酶以防治肿瘤的问题上,针对不同人群、不同疾病状况及不同用药方案可能需采取抑制或诱导的不同策略。 相似文献
11.
Courtney M. Krest Elizabeth L. Onderko Timothy H. Yosca Julio C. Calixto Richard F. Karp Jovan Livada Jonathan Rittle Michael T. Green 《The Journal of biological chemistry》2013,288(24):17074-17081
Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. 相似文献
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The cytochrome P450 mono-oxygenase system represents a major defence against chemical challenge from the environment, constituting part of an adaptive response mounted by an organism following exposure to harmful agents. Cytochrome P450s are also able to catalyse the activation of compounds to toxic products, and participate in a variety of essential 'housekeeping' functions, such as biosynthesis of steroid hormones and fatty acid oxidation. It is clear that the modulation of expression of these enzymes can have a significant effect on chemical toxicity, carcinogenicity and mutagenicity. The concept of cancer chemoprevention, i.e. the administration of a (non-toxic) chemical or dietary component in order to prevent neoplastic disease or to inhibit its progression, is an attractive one. Despite this, relatively little work has been done to characterize the ability of putative chemopreventive agents to modulate P450 expression, or to understand the interaction between P450s and chemopreventive agents. Before chemopreventive treatment can become a reality, it is essential that this complex issue is addressed; for instance, it is likely that any single chemopreventive agent will induce more than one P450 isoenzyme, and while altered expression of a particular P450 may attenuate the effects of one toxic agent, the effects of others might well be potentiated. Our laboratory has created a transgenic mouse line in which the rat CYP1A1 promoter drives expression of the beta-galactosidase gene. These mice can be used to define which compounds act via the Ah receptor, in which tissues, and at which stage of development. We are currently developing another mouse line in which beta1-galactosidase expression is controlled by the mouse GstA1 promoter, allowing us to define the role of the antioxidant responsive element in the action of chemopreventive agents. Finally, using cre-loxP transgenic technology, we have generated a mouse line in which P450 reductase can be deleted in a conditional, i.e. tissue-specific, manner, permitting us to investigate the role of P450s in chemoprevention in a more defined manner. 相似文献
15.
A G Buchatskii A A Kazachenko KYuAlexandrov
The pattern suggested for the structure-function superfamily of cytochromes P450 is composed by combining the conserved amino acid motifs. The sizes of P450 cytochromes were estimated according to their length. The empirical coefficients reflecting the peculiarities of the primary structure of these enzymes are calculated. We propose an approach for determining novel proteins sequences to the mentioned superfamily on the ground of the complex of these parameters. A number of the hypothetical proteins from the international databases is related to the cytochromes P450 by means of our pattern. 相似文献
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链霉菌中存在大量的细胞色素P450,它们在链霉菌次生代谢产物的生物合成和外来化学物质代谢过程中发挥了重要作用.本文综述了链霉菌中发现的细胞色素P450及其功能的研究进展,分析了存在的问题和研究应用前景. 相似文献
18.
Cytochrome P450 oxygenases of Taxol biosynthesis 总被引：3，自引：0，他引：3
19.
Cytochrome P450 oxygenases of monoterpene metabolism 总被引：2，自引：0，他引：2
The cytochrome P450 monoterpene oxygenases are largely responsible for imparting structural and functional diversity to this family of natural products. In most cases, cytochrome P450-mediated allylic hydroxylation of a parental monoterpene olefin leads to a series of redox transformations and conjugation reactions which yield a family of structurally related derivatives and isomers. An overview is provided of the extant monoterpene oxygenases, with examples mainly from the mint (Lamiaceae) family of essential oil plants, and, where possible, information on the structure, mechanism, localization and regulation of these enzymes is described. The review concludes with a brief assessment of biotechnological applications and a view to future research in this area. 相似文献