Effect of homomeric P450-P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.     

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.     

The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase.

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.     

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.     

Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.     

Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (A1) of a new P450 gene family (LII).     

Mitochondrial P450s

Cytochrome P450 was first found in the microsomes from animal tissues, and then the presence of P450 in mitochondria was reported for the steroidogenic organs, adrenal gland and gonads. Three forms of mitochondrial P450 (11A, 11B1, and 11B2) were purified from these organs and their functions in steroid hormone biosynthesis were confirmed. Later studies showed the presence of several other forms of P450 (24A, 27A, 27B, and 27C) in the mitochondria of various non-steroidogenic organs including liver and kidney. These mitochondrial P450s were found to participate in the biosynthesis of bile acids from cholesterol in the liver, and the metabolic activation of Vitamin D3 to its active form, 1,25-dihydroxyvitamin D3, in the liver and the kidney. In contrast to the "drug-metabolizing" P450s in microsomes, most mitochondrial P450s show high specificity to their endogenous substrates, and have negligible activity towards xenobiotic compounds. In contrast to these established roles of mitochondrial P450s in the metabolism of endogenous substrates, the metabolism of xenobiotic chemicals by P450-catalyzed reactions in mitochondria has long been a subject of controversy. It is now known that all P450s in eukaryotic organisms are coded by nuclear genes, and the nascent peptides of various forms of P450 synthesized by cytoplasmic ribosomes are targeted to either endoplasmic reticulum (ER) or mitochondria depending on the ER-targeting sequence or the mitochondria-targeting sequence present in their amino-terminal portion. However, the presence of some microsome-type P450s in the mitochondria from various animal tissues including liver and brain has been reported. Possible mechanisms of intracellular sorting of some microsome-type P450s to mitochondria have been proposed, although physiological significance of the contribution of P450s in mitochondria to the metabolism of xenobiotic chemicals in animal tissues is still elusive.     

植物细胞色素P450

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。     

Human cytochromes P450

The cytochrome P450 proteins (CYPs) are a family of haem proteins resulting from expression of a gene super-family that currently contains around 1000 members in species ranging from bacteria through to plants and animals. In humans, about 40 different CYPs are present and these play critical roles by catalyzing reactions in: (a) the metabolism of drugs, environmental pollutants and other xenobiotics; (b) the biosynthesis of steroid hormones; (c) the oxidation of unsaturated fatty acids to intracellular messengers; and (d) the stereo- and regio-specific metabolism of fat-soluble vitamins. This review deals with aspects of cytochrome P450s of relevance to human physiology, biochemistry, pharmacology and medicine. Topics reviewed include: pharmacogenetics of CYPs, induction and inhibition of these haem proteins, their role in metabolism of endogenous compounds such as steroids and eicosanoids, the effect of disease on CYP function, CYPs and cancer, and CYPs as targets of antibodies in immune-mediated diseases.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Structural characterization of n-butyl-isocyanide complexes of cytochromes P450nor and P450cam

Alkyl-isocyanides are able to bind to both ferric and ferrous iron of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra. While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm. The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme. To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm. Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively. The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined. The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam [d(Fe-C) = 1.86 A, angleFe-C-N = 159 degrees ] versus P450nor [d(Fe-C) = 1.85 A, angleFe-C-N = 175 degrees ]. Another difference in the structures was the chemical environment of the heme pocket. In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules. On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.     

Origins of P450 diversity

The P450 enzymes maintain a conserved P450 fold despite a considerable variation in sequence. The P450 family even includes proteins that lack the single conserved cysteine and are therefore no longer haem-thiolate proteins. The mechanisms of successive gene duplications leading to large families in plants and animals are well established. Comparisons of P450 CYP gene clusters in related species illustrate the rapid changes in CYPome sizes. Examples of CYP copy number variation with effects on fitness are emerging, and these provide an opportunity to study the proximal causes of duplication or pseudogenization. Birth and death models can explain the proliferation of CYP genes that is amply illustrated by the sequence of every new genome. Thus, the distribution of P450 diversity within the CYPome of plants and animals, a few families with many genes (P450 blooms) and many families with few genes, follows similar power laws in both groups. A closer look at some families with few genes shows that these, often single member families, are not stable during evolution. The enzymatic prowess of P450 may predispose them to switch back and forth between metabolism of critical structural or signal molecules and metabolism dedicated to environmental response.     

绿盲蝽P450基因的克隆及增效醚对P450酶活性的抑制作用

为探讨P450介导的绿盲蝽Apolygus lucorum(Meyer-Dür)抗药性机制,合理使用杀虫药剂,本研究通过活体和离体抑制实验发现,增效醚(PBO)对绿盲蝽P450酶活性有显著的抑制作用:在处理时长为24h时,P450酶活性由未处理时的12.02pmol/min/mgPro.下降至1.63pmol/min/mgPro.,PBO对P450酶的抑制中浓度为0.256mmol/L。生物测定结果表明,PBO对三氟氯氰菊酯具有显著增效作用,增效7.2倍,而对吡虫啉、灭多威、马拉硫磷无显著增效作用。利用RT-PCR及RACE技术对绿盲蝽P450基因进行克隆,获得了2条CYP4家族基因,全长均为1631bp,含有完整的开放阅读框,编码501个氨基酸;序列比对表明这是一对等位基因,含有CYP4家族所有保守特征序列;同源性比较及系统发育分析显示这2个基因编码的氨基酸序列与褐飞虱Nilaparvata lugens CYP4CE1亲缘关系最近,同源性分别为41.5%和41.1%。     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Keeping the spotlight on cytochrome P450

This review describes the recent advances utilizing photosensitizers and visible light to harness the synthetic potential of P450 enzymes. The structures of the photosensitizers investigated to date are first presented along with their photophysical and redox properties. Functional photosensitizers range from organic and inorganic complexes to nanomaterials as well as the biological photosystem I complex. The focus is then on the three distinct approaches that have emerged for the activation of P450 enzymes. The first approach utilizes the in situ generation of reactive oxygen species entering the P450 mechanism via the peroxide shunt pathway. The other two approaches are sustained by electron injections into catalytically competent heme domains either facilitated by redox partners or through direct heme domain reduction. Achievements as well as pitfalls of each approach are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Tissue-specific induction of the carcinogen inducible cytochrome P450 isoform, P450IAI, in colonic epithelium

The epithelial cells of the gastrointestinal tract have the capacity to engage in biotransformation of ingested chemicals. A principal component of phase I metabolism of xenobiotics is the family of hemeproteins referred to as cytochrome(s) P450. The presence of cytochrome P450 isoforms was examined by Western blot analysis in the epithelial cells of the colon and proximal small intestine of male rats following oral administration with either beta-naphthoflavone or phenobarbital. The appearance of beta-naphthoflavone-inducible cytochrome P450IAI was observed in the colon and small intestine. The appearance of this cytochrome P450 isoform was concurrent with increases (up to 150-fold) in cytochrome P450-related O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin in both tissues. Following administration of phenobarbital, cytochrome P450IIBI was identified immunochemically in the small intestine. However, this isozyme could not be detected in colon. These data suggest that the epithelial cells of the proximal small intestine respond to beta-naphthoflavone and phenobarbital in a manner similar to the liver, whereas colonic epithelial cells may have a greater capacity to respond to P450IAI-type inducers such as beta-naphthoflavone. Evidence exists that differences in cytochrome P450 isozyme composition can affect the ultimate metabolic fate of ingested chemicals, including carcinogens, and thus a role for colonic P450-dependent monooxygenase activity in the biogenesis of cancer in this tumor-susceptible tissue is suggested.