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.     

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.     

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.     

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.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

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.     

Putidaredoxin-cytochrome P450cam interaction

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.     

Cytochrome P450 biosensors-a review

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.     

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.     

Moonlighting cytochrome P450 monooxygenases

Recently, cytochrome P450 170A1 (CYP170A1) has been found to be a bifunctional protein, which catalyzes both monooxygenase activity and terpene synthase activity by two distinct active sites in the well-established P450 protein structure. Therefore, CYP170A1 is identified clearly as a moonlighting protein. The known activities of a small number of the 13,000 members of the P450 superfamily fall into two general classes: promiscuous enzymes that are not considered as moonlighting and forms that participate in biosynthesis of endogenous compounds, such as steroids, vitamins and play different roles in different tissues, sometimes being moonlighting enzymes. Here, we review examples of moonlighting P450, which add to our understanding of the large CYP superfamily.     

Plant NADPH-cytochrome P450 oxidoreductases

NADPH-cytochrome P450 oxidoreductase (CPR) serves as the electron donor to almost all eukaryotic cytochromes P450. It belongs to a small family of diflavin proteins and is built of cofactor binding domains with high structural homology to those of bacterial flavodoxins and to ferredoxin-NADP⁺ oxidoreductases. CPR shuttles electrons from NADPH through the FAD and FMN-cofactors into the central heme-group of the P450s. Mobile domains in CPR are essential for electron transfer between FAD and FMN and for P450 interaction. Blast searches identified 54 full-length gene sequences encoding CPR derived from a total of 35 different plant species. CPRs from vascular plants cluster into two major phylogenetic groups. Depending on the species, plants contain one, two or three paralogs of which one is inducible. The nature of the CPR–P450 interacting domains is well conserved as demonstrated by the ability of CPRs from different species or even from different kingdoms to at least partially complement each other functionally. This makes CPR an ideal bio-brick in synthetic biology approaches to re-design or develop entirely different combinations of existing biological systems to gain improved or completely altered functionalities based on the “share your parts” principle.