Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450

Cytochrome P450-dependent oxidation of arachidonic acid was studied in liver microsomes from normal fed, protein-energy malnourished, and refed rats. The overall rate of arachidonic acid oxidation was very similar in microsomes from the three groups, but microsomes from malnourished rats showed a higher turnover rate than microsomes from normal fed and refed rats. The regiospecificity of cytochrome P450 oxidation of arachidonic acid was drastically altered by the animal nutritional status. Thus, protein-energy malnutrition results in a clear stimulation of total omega and omega-1 hydroxylation, concomitant with a marked decrease in olefin epoxidation and allyllic oxidations. These changes, as well as the documented biological activity of some of the cytochrome P450 arachidonate metabolites, suggest that protein-energy deficiency might help to select P450 isozymes which are probably involved in key monooxygenation reactions of physiological substrates.     

omega-1 and omega-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6

Incubation of prostaglandin E1 (PGE1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at omega-2, omega-1, and omega carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the omega-1 and omega-2 hydroxylation of PGE1 and PGE2. Additionally, a small amount of product with chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b5 into the reconstituted system did not enhance the rate of PGE1 hydroxylation and had no effect on the ratio of products formed. The Km value for the omega-1 and omega-2 hydroxylation of PGE1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 microM; the Vmax's (nmol product min-1 nmol P-450-1) were 2.1 and 1.1 for the omega-1 and omega-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the omega-2 hydroxylation of PGEs observed in rabbit liver microsomes.     

High pressure liquid chromatography methods for separation of omega- and (omega-1)-hydroxy fatty acids: their applications to microsomal fatty acid omega-oxidation

Fatty acids (C12-C18) and their omega- and (omega-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% (v/v) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the omega- and (omega-1)-hydroxy derivatives are 14-20 and 24-29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a muBondapak C18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265-272) for separation of methyl esters of fatty acids and their omega- and (omega-1)-hydroxy derivatives. In the latter method, however, the retention times of omega- and (omega-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid omega oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.     

Production of hydroxy-fatty acid derivatives from waste oil by <Emphasis Type="Italic">Escherichia coli</Emphasis> cells producing fungal cytochrome P450foxy

Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7-16 carbon atoms to their omega-1 to omega-3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to omega-1 to omega-3 hydroxy decanoic acids in the order of omega-1>omega-2>omega-3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of omega-1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry.     

Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA) contribute to the regulation of cardiovascular function. CYP enzymes also accept EPA and DHA to yield more potent vasodilatory and potentially anti-arrhythmic metabolites, suggesting that the endogenous CYP-eicosanoid profile can be favorably shifted by dietary omega-3 fatty acids. To test this hypothesis, 20 healthy volunteers were treated with an EPA/DHA supplement and analyzed for concomitant changes in the circulatory and urinary levels of AA-, EPA-, and DHA-derived metabolites produced by the cyclooxygenase-, lipoxygenase (LOX)-, and CYP-dependent pathways. Raising the Omega-3 Index from about four to eight primarily resulted in a large increase of EPA-derived CYP-dependent epoxy-metabolites followed by increases of EPA- and DHA-derived LOX-dependent monohydroxy-metabolites including the precursors of the resolvin E and D families; resolvins themselves were not detected. The metabolite/precursor fatty acid ratios indicated that CYP epoxygenases metabolized EPA with an 8.6-fold higher efficiency and DHA with a 2.2-fold higher efficiency than AA. Effects on leukotriene, prostaglandin E, prostacyclin, and thromboxane formation remained rather weak. We propose that CYP-dependent epoxy-metabolites of EPA and DHA may function as mediators of the vasodilatory and cardioprotective effects of omega-3 fatty acids and could serve as biomarkers in clinical studies investigating the cardiovascular effects of EPA/DHA supplementation.     

Regiospecific hydroxylation of lauric acid at the (omega-1) position by hepatic and kidney microsomal cytochromes P-450 from rainbow trout

Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout.     

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions.

The soluble P450 isolated from Bacillus megaterium (the product of the CYP 102 gene) (P450BM-3) is a catalytically self-sufficient fatty acid hydroxylase which converts lauric, myristic, and palmitic acids to omega-1, omega-2, and omega-3 hydroxy analogs. The percentage distribution of the regioisomers depends on the substrate chain length. Lauric and myristic acids were preferentially metabolized to their omega-1 hydroxy counterparts while no hydroxylation occurred when capric acid was used as the substrate. Palmitic acid, when present at concentrations greater than the concentration of oxygen in the reaction medium (greater than 250 microM), was hydroxylated to its omega-1, omega-2, and omega-3 hydroxy analogs, with the percentage distribution of the regioisomers being 21:44:35, respectively. No omega hydroxylation of any of the fatty acids was detected. When the concentration of palmitic acid was less than the concentration of oxygen in the reaction mixture, it was noted that a number of additional products were formed. Under these conditions, unlike lauric and myristic acids, it was observed that palmitic acid was first converted to its monohydroxy isomers which were subsequently metabolized to a mixture of 14-ketohexadecanoic, 15-ketohexadecanoic, 13-hydroxy-14-ketohexadecanoic, 14-hydroxy-15-ketohexadecanoic, and 13,14-dihydroxyhexadecanoic acids with a relative distribution of 8:2:40:30:20, respectively. Thus, P450BM-3 is able not only to monohydroxylate a variety of fatty acids but also to further metabolize some of these primary metabolites to secondary and tertiary products. The present paper characterizes the products formed during the sequential hydroxylation of palmitic acid and proposes reaction pathways to explain these results.     

Cloning and expression of three rabbit kidney cDNAs encoding lauric acid omega-hydroxylases

cDNAs encoding three cytochrome P-450 enzymes were cloned from a rabbit kidney cDNA library. These three cDNAs exhibit greater than 90% nucleotide sequence identity across the coding region. This degree of sequence identity is also seen with P450IVA4, an enzyme that catalyzes the omega-hydroxylation of prostaglandins and that is elevated during pregnancy and induced by progesterone in rabbit lung. The 3' untranslated regions of the three cDNAs display very little sequence identity, suggesting that they are the products of distinct genes. The predicted amino acid sequences derived from each cDNA and for P450IVA4 exhibit about 85% identity. Each cDNA was inserted into an expression vector for transient transfection of COS-1 cells. The transfected cells each expressed a protein recognized by antibodies to P450IVA4. Microsomes isolated from the cells transfected with each cDNA efficiently catalyzed the omega-hydroxylation of lauric acid with rates that greatly exceed that catalyzed by microsomes isolated from the host cell line. One of the cDNAs encodes an enzyme that omega-hydroxylates prostaglandin A1; however, the specific activity was 2 orders of magnitude lower than that for lauric acid. Our results indicate that the substrate selectivity of the kidney P-450s encoded by these cDNAs is distinct from that of the lung P450IVA4 and that multiple enzymes comprise P-450 class IVA in the rabbit.     

Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450

We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position.     

Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.     

Regiospecificity of human cytochrome P450 1A1-mediated oxidations: the role of steric effects

Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobiotic benzo[a]pyrene (B[a]P) and endogenous fatty acid precursors of prostaglandins, such as arachidonic acid (AA) and eicosapentaenoic acid (EA). We have investigated the extent to which enzyme-substrate interactions govern regio- and stereoselectivity of oxidation of these compounds by using docking and molecular dynamics (MD) simulations to examine the likelihood of substrate oxidation at various sites. Due to structural differences between the substrates analyzed, B[a]P and its diols (planar, rigid), and the fatty acids AA and EA (long, flexible), different docking strategies were required. B[a]P, B[a]P-7,8-diols, (+) 7S,8S- and (-) 7R,8R-diols, were docked into the active site of a homology model of P450 1A1 using an automated routine, Affinity (Accelrys, San Diego, CA). AA and EA, on the other hand, required a series of restrained MD simulations to obtain a variety of productive binding modes. All complexes were evaluated by MD-based in silico site scoring to predict product profiles based on certain geometric criteria, such as angle and distance of a given substrate atom from the ferryl oxygen. For all substrates studied, the in vitro profiles were generally reflected by the in silico scores, which suggests that steric factors play a key role in determining regiospecificity in P450 1A1-mediated oxidations. We have also shown that molecular dynamics simulations may be very useful in determination of product profiles for structurally diverse substrates of P450 enzymes.     

Differential tissue-specific expression and induction of cytochrome P450IVA1 and acyl-CoA oxidase.

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.     

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.     

Purification and characterization of two forms of cytochrome P-450 with omega-hydroxylase activities toward prostaglandin A and fatty acids from rabbit liver microsomes

Two forms of cytochrome P-450 (P-450), designated as P-450LPGA omega 1 and P-450LPGA omega 2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LPGA omega 1 and P-450LPGA omega 2 were found to have apparent molecular weights of 52,500 and 53,000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the omega-hydroxylation of prostaglandins A1 (PGA1) and A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFSGFLQAxGLLGLLL) of P-450LPGA omega 1 and P-450LPGA omega 2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin omega-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPGA omega 2 recognized a 52,000-53,000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids

Cytochrome P-450LA omega purified from clofibrate-induced rat liver oxidizes lauric acid to 11- and 12-hydroxydodecanoic acid in approximately a 1:17 ratio at a rate of 20 nmol/nmol P-450/min. In contrast, cytochrome P-450b oxidizes lauric acid much more slowly (0.5 nmol/nmol P-450/min) to an 8:1 mixture of the same metabolites. Western blot analysis indicates that P-450LA omega accounts for 1-2 and 16-30%, respectively, of the total cytochrome P-450 in uninduced and clofibrate-induced rat liver. Cytochrome b5 increases the efficiency of omega-hydroxylation but not the rate of catalytic turnover. Incubation of the enzyme with 10-undecynoic acid (10-UDYA) results in loss of approximately 45% of the enzymatic activity but none of the enzyme chromophore. Approximately 1 mol of 1,11-undecandioic acid is produced per mole of inactivated enzyme. This extraordinary inactivation efficiency is confirmed by NADPH consumption studies. Approximately 0.5 equivalents of label are covalently bound to the enzyme when it is incubated with 14C-labeled 10-UDYA. 11-Dodecenoic acid appears not to be a substrate for cytochrome P-450LA omega but is oxidized, presumably by a contaminating isozyme, to a 10:1 mixture of 11,12-epoxydodecanoic acid and 12-oxododecanoic acid. The results suggest the presence of two closely related P-450LA omega enzymes, only one of which is susceptible to inactivation by 10-UDYA. They also indicate that cytochrome P-450LA omega has a highly structured active site that sterically suppresses omega-1-hydroxylation in order to deliver the oxygen to the thermodynamically disfavored terminal carbon. Protein rather than heme alkylation follows from this reaction regiospecificity.     

Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system.

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (omega-1 approximately omega-3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane-bound native protein. The results are consistent with our notion that the native protein is targeted to the membrane by a post-translational modification mechanism. We also discovered that P450foxy could use shorter saturated fatty acid chains (C9 and C10) as a substrate. The regiospecificity (omega-1 approximately omega-3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates. Steady state kinetic studies showed that the kcat values for all substrates tested (C9-C16) were of the same magnitude (1200-1800 min-1), whereas the catalytic efficiency (kcat/Km) was higher for longer fatty acids. Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length. This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified. Kinetic data were consistent with the inhibition due to binding of the second substrate. We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding.     

Microsomal omega-hydroxylated metabolites of N-arachidonoyl dopamine are active at recombinant human TRPV1 receptors

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that modulates signal transduction in neuronal and immune pathways. NADA activates the non-selective cation channel, transient receptor potential vanilloid type 1 (TRPV(1)) and cannabinoid receptor 1. That NADA is comprised of an arachidonic acid (AA) backbone suggests that it may be metabolized through many of the enzymes that act upon AA such as the other AA-derived signaling lipids, the endogenous cannabinoids. To investigate the metabolism of NADA through the cytochrome P450 (CYP450) metabolic pathway, we studied the in vitro rat liver microsomal production of hydroxylated metabolites and their activity at recombinant human TRPV(1) receptors. We showed that following microsomal activation in the presence of NADA, omega and (omega-1) hydroxylated metabolites (19- and 20-HETE-DA) were formed. These metabolites were active at recombinant human TRPV(1) receptors, inducing a dose-dependent calcium influx. Both metabolites exhibited lower potency compared to NADA. We conclude that CYP450 enzymes are capable of metabolizing this signaling lipid forming a larger family of potential neuromodulators.