Cytochrome P450-dependent metabolism of arachidonic acid.

The cytochrome P450-dependent metabolism of arachidonic acid, the mechanisms of regulation of stereo- and regiospecificity by cytochrome P450 isoenzymes, and the biological relevance of metabolites of the arachidonic acid cascade is discussed in this review.     

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.     

Cytochrome P450 pathways of arachidonic acid metabolism

Cytochrome P450s metabolize arachidonic acid to hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids. These eicosanoids are formed in a tissue and cell-specific manner and have numerous biological functions. Of major interest are the opposing actions of hydroxyeicosatetraenoic and epoxyeicosatrienoic acids within the vasculature. Regio- and stereoisomeric epoxyeicosatrienoic acids have potent vasodilatory properties while 20-hydroxyeicosatetraenoic acid is a potent vasoconstrictor. Both effects are mediated through actions on large-conductance Ca2+-activated K+ channels. Cytochrome P450-derived eicosanoids are also important in the regulation of ion transport, and have recently been shown to influence a number of fundamental biological processes including cellular proliferation, apoptosis, inflammation, and hemostasis. The formation of these functionally relevant eicosanoids is tightly controlled by the expression and activity of the cytochrome P450 epoxygenases and hydroxylases. In addition, soluble epoxide hydrolase catalyzes the hydrolysis of epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids, and the activity of this enzyme is a critical determinant of tissue epoxyeicosatrienoic and dihydroxyeicosatrienoic acid levels. The intracellular balance between epoxyeicosatrienoic, dihydroxyeicosatrienoic and hydroxyeicosatetraenoic acids influences the biological response to these eicosanoids and alterations in their levels have recently been associated with certain pathological conditions. The involvement of the cytochrome P450-derived eicosanoids in a wide array of biological functions and the observation that levels are altered in pathological conditions suggest that the enzymes involved in the formation and degradation of these fatty acids may be novel therapeutic targets.     

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.     

Cytochrome P450 monooxygenases: perspectives for synthetic application

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.     

P450-based porous silicon biosensor for arachidonic acid detection

A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 μM arachidonic acid in a dynamic range of 10-200 μM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 μM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 μM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.     

Purification and properties of a cytochrome P450 arachidonic acid epoxygenase from rabbit renal cortex.

A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.     

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

INTRODUCTION It is well known that arachidonic acid (AA) exists ex- tensively in eukaryotic cells and it is metabolized by cycloxygenases and lipoxygenases to produce prostag- landins (PGs) and hydroperoxyeicosatetraenoic acid(HPETE) [1-3]. In the 1980’s…     

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.     

Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.     

Cytochrome P450 monooxygenases of DIBOA biosynthesis: specificity and conservation among grasses.

DIBOA and DIMBOA are secondary metabolites of grasses which function as natural pesticides. The four maize genes BX2 through BX5 encode cytochrome P450-dependent monooxygenases that catalyse four consecutive reactions in the biosynthesis of these secondary products. Although BX2-BX5 share significant sequence homology, the four enzymes have evolved into specific enzymes each catalysing predominantly only one reaction in the pathway. In addition to these natural reactions, BX3 hydroxylates 1,4-benzoxazin-3-one and BX2 shows pCMA demethylase activity. With respect to DIBOA biosynthesis, identical enzymatic reactions have been found in rye as compared to maize, indicating early evolution of the P450 enzymes in the grasses.     

Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospeci?c oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.     

Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase

The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.     

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.     

Ordered chimerogenesis applied to CYP2B P450 enzymes

BackgroundStructural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.MethodsA series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.ResultsMost of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels.ConclusionsSome sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.General significanceThe result provides a new basis for determining the structural elements that control functions in complex enzymes.