Omega-1 Hydroxylation Research Articles

Fungal cytochrome P450 monooxygenases of Fusarium oxysporum for the synthesis of ω-hydroxy fatty acids in engineered Saccharomyces cerevisiae.

BackgroundOmega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom.ResultsTo gain a deep insight on the biochemical role of fungal P450s towards the production of omega hydroxy fatty acids, two cytochrome P450 monooxygenases from Fusarium oxysporum (FoCYP), FoCYP539A7 and FoCYP655C2; were identified, cloned, and heterologously expressed in Saccharomyces cerevisiae. For the efficient production of ω-OHFAs, the S. cerevisiae was engineered to disrupt the acyl-CoA oxidase enzyme and the β-oxidation pathway inactivated (ΔPox1) S. cerevisiae mutant was generated. To elucidate the significance of the interaction of redox mechanism, FoCYPs were reconstituted with the heterologous and homologous reductase systems - S. cerevisiae CPR (ScCPR) and F. oxysporum CPR (FoCPR). To further improve the yield, the effect of pH was analyzed and the homologous FoCYP-FoCPR system efficiently hydroxylated caprylic acid, capric acid and lauric acid into their respective ω-hydroxy fatty acids with 56%, 79% and 67% conversion. Furthermore, based on computational simulations, we identified the key residues (Asn106 of FoCYP539A7 and Arg235 of FoCYP655C2) responsible for the recognition of fatty acids and demonstrated the structural insights of the active site of FoCYPs.ConclusionFungal CYP monooxygenases, FoCYP539A7 and FoCYP655C2 with its homologous redox partner, FoCPR constitutes a promising catalyst due to its high regio- and stereo-selectivity in the hydroxylation of fatty acids and in the substantial production of industrially valuable ω-hydroxy fatty acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0228-2) contains supplementary material, which is available to authorized users.

FACTORS AFFECTING LIPID OXIDATION IN BREAST AND THIGH MUSCLE FROM CHICKEN, TURKEY AND DUCK

ABSTRACT Lipid oxidation occurred rapidly in turkey muscle, intermediate in duck and slowest in chicken. pH was lowest in turkey muscle. Chicken muscle had a lower content of polyunsaturated fatty acids compared with turkey and duck muscles. The aqueous fraction of duck breast inhibited hemoglobin-mediated lipid oxidation in washed muscle more effectively than aqueous fractions from turkey and chicken muscle. α-Tocopherol content was highest in duck muscle, intermediate in chicken and lowest in turkey. Depletion of tocopherols during frozen storage was more rapid in turkey and duck compared with chicken. It was thought that the elevated tocopherol level in chicken muscle may be caused by less efficient catabolism via the omega hydroxylation pathway. However, tocopherol hydroxylase activity was similar in chicken compared with turkey liver microsomes. Heme pigment content was around sixfold higher in duck breast compared with chicken and turkey breast. Duck thigh had especially elevated pH. PRACTICAL APPLICATIONS This work describes a number of factors that explain the wide variation in oxidative stability (chicken > duck > turkey) when comparing muscle tissues from the three avian species. These factors include muscle pH, concentration of heme pigments, fatty acid unsaturation, inhibitors of lipid oxidation in the aqueous fraction of the muscle, tocopherol content in lipid phases and depletion rates of tocopherol. These factors should be considered when developing strategies to inhibit lipid oxidation in muscle foods. The relatively high content of α-tocopherol in chicken muscle compared with turkey should be a subject of further research to better understand the mechanisms by which certain animal species preferentially deposit the molecule into muscle.

Structures of Human Cytochrome P-450 2E1: INSIGHTS INTO THE BINDING OF INHIBITORS AND BOTH SMALL MOLECULAR WEIGHT AND FATTY ACID SUBSTRATES

Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.

Resolvin E1 Metabolome in Local Inactivation during Inflammation-Resolution

Resolvin E1 (RvE1; 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a potent anti-inflammatory and proresolving mediator derived from the omega-3 eicosapentaenoic acid. In this study, we report the RvE1 metabolome, namely, the metabolic products derived from RvE1. RvE1 was converted to several novel products by human polymorphonuclear leukocytes and whole blood as well as in murine inflammatory exudates, spleen, kidney, and liver. The potential activity of each of the newly identified products was directly compared with that of RvE1. The new RvE1 products elucidated included 19-hydroxy-RvE1, 20-carboxy-RvE1, and 10,11-dihydro-RvE1. Metabolomic profiles of RvE1 were species-, tissue-, and cell type-specific. Direct comparisons of the bioactions between isolated RvE1 metabolic products indicated that 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 displayed reduced bioactivity in vivo. At concentrations as low as 1 nM, RvE1 enhanced macrophage phagocytosis, a proresolving activity that was reduced by metabolic inactivation. These results document novel metabolic products of RvE1 that impact its actions and that both omega-1 hydroxylation and reduction of conjugated double bonds in RvE1 are new pathways of four main routes of RvE1 metabolism in mammalian tissues. Together, these findings indicate that, during inflammation and its controlled resolution, specific tissues inactivate proresolving signals, i.e., RvE1, to permit the coordinated return to homeostasis. Moreover, the RvE1 metabolome may serve as a biomarker of these processes.

Covalent heme binding to CYP4B1 via Glu310 and a carbocation porphyrin intermediate.

Recently we found that CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their prosthetic heme group through an ester linkage. In the current study, we mutated a conserved CYP4 I-helix residue, E310 in rabbit CYP4B1, to glycine, alanine, and aspartate to examine the effect of these mutations on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells and were isolated as a mixture of monomeric and dimeric forms as determined by LC/ESI-MS of the intact proteins. Rates of metabolism decreased in the order E310 > A310 >> G310 > D310, with the A310 and G310 mutants exhibiting alterations in regioselectivity for omega-1 and omega-2 hydroxylation of lauric acid, respectively. In marked contrast to the wild-type E310 enzyme, the G310, A310, and D310 mutants did not bind heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H(2)(18)O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data show (i) that E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; and (iii) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin.

Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.

CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K(m) value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4'-hydroxylation of diclofenac, and 3-fold higher for the omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V(max)/K(m), ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.

Reconstitution of the Enzymatic Activities of Cytochrome P450s Using Recombinant Flavocytochromes Containing Rat Cytochrome b5 Fused to NADPH–Cytochrome P450 Reductase with Various Membrane-Binding Segments

The role of the hydrophobic membrane-binding segments of NADPH–cytochrome P450 reductase (CPR) and cytochrome b5 remain undefined. We have expressed four different recombinant flavocytochromes containing b5 linked to CPR with different hydrophobic segments as linkers. These fusion proteins have been expressed in Escherichia coli and purified and some of their physical properties and electron transfer activities described in the accompanying paper. Of interest is the presence of internal “membrane-binding” hydrophobic segments in these flavocytochromes. This paper describes the ability of these flavocytochromes to reconstitute in vitro two P450 activities that have been reported to be stimulated by the addition of b5 (the 17,20-lyase activity of CYP17A and the 6β hydroxylation of testosterone catalyzed by CYP3A4) and two P450 reactions that do not respond to the presence of b5 (the 17α-hydroxylation of progesterone catalyzed by CYP17A and the omega hydroxylation of lauric acid catalyzed by CYP4A1). The present study shows that a hydrophobic “membrane-binding” segment must be present in the artificial flavocytochromes in order to successfully reconstitute in vitro hydroxylation activities with P450s. Differences in the effectiveness of the different flavocytochromes to reconstitute enzymatic activities depends on the P450 tested and the nature of the hydrophobic linker segment present in the purified recombinant flavocytochromes. The hypothesis is proposed that differences in the surface topology of a P450 may dictate differences in their docking with the CPR or b5 component of a fusion protein, resulting in differences in the rates of electron transfer to the P450.

The Catalytic Site of Cytochrome P4504A11 (CYP4A11) and Its L131F Mutant

CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.

Hydroxylation of carbon atoms of the alkyl chain of symmetrical N-nitrosodialkylamines by rat liver microsomes.

Liver microsomal preparations from control and treated rats (cytochromes P450 1A, 2B, 3A and 2E1-induced) metabolized at variable metabolic rates three nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines) into aldehydes and hydroxy-nitrosamines. The longer the alkyl chain, the smaller was the metabolic rate of the alpha-hydroxylation of alkyl chain yielding aldehyde and the greater was the metabolic rate of the corresponding (omega-1)-hydroxyl metabolite formation. Thus, the (omega-1) hydroxylation of the alkyl chain was the major metabolic pathway of N-nitrosodiamylamine (NDAA) so far as it represented 22-fold the alpha-hydroxylation. The balance between beta to omega hydroxylation and alpha-hydroxylation depends upon the alkyl chain length and also on specific P450 isoform induction.

Effects of Steric Bulk and Conformational Rigidity on Fatty Acid Omega Hydroxylation by a Cytochrome P450 4A1 Fusion Protein

The action of cytochrome P450 4A1 (CYP4A1) on fatty acid substrates is characterized by a pronounced regioselectivity for ω-hydroxylation. To elucidate the chemical basis of this specificity we probed the active site of a CYP4A1 fusion protein (f4A1) with bulky and/or rigid analogs of lauric acid, the optimum natural substrate for f4A1 and CYP4A1. f4A1 efficiently ω-hydroxylates lauric acid, epoxidizes 11-dodecenoic acid, and oxidizes 11-dodecynoic acid to 1,12-dodecanedioic acid. Medium length fatty acids having ω-terminal groups as large ast-butyl orm-tolyloxy bind tightly to f4A1 as Type I ligands and are efficiently hydroxylated on their methyl termini. ω-Phenylnonanoic acid also induces a Type I binding spectrum (Ks= 0.77 μm) but fails to undergo hydroxylation and strongly inhibits lauric acid hydroxylation by f4A1. Slightly shorter acids such as ω-phenyloctanoic acid, naproxen, and ibuprofen also strongly inhibit lauric acid hydroxylation but do not induce a Type I spectrum and do not undergo hydroxylation. Like 10-methoxydecanoic acid, the rigid and rod-like 4′-methoxy-4-biphenylcarboxylic acid is O-demethylated by f4A1, which also ω-hydroxylatesm- andp-heptyloxybenzoic acids but notm- orp-amyloxyhydrocinnamic acids. The histamine antagonist cimetidine and the peroxisome proliferator perfluorodecanoic acid are both potent inhibitors of f4A1. Thus the active site of f4A1 is quite tolerant of steric bulk and rigidity around the heme region and the polar group recognition site, but perhaps less so in the midchain region. Although CYP4A enzymes are not usually regarded as “drug metabolizing P450s,” the fact that commonly used therapeutic agents strongly inhibit lauric acid ω-hydroxylation by f4A1 as well as liver microsomes from clofibrate-induded rats suggests these and related agents could potentially interfere with the contribution of CYP4A enzymes to the metabolism of endogenous lipids.

Cytochrome P450-dependent oxidation of fatty acids.

Cytochrome P450-dependent monooxygenases from plants catalyse in-chain and omega hydroxylation as well as epoxidation of medium- and long-chain fatty acids. Recent research efforts have clarified that there are multiple forms of cytochrome P450 involved in these reactions, each of which possesses distinguishable substrate specificity. The biological roles of these distinct P450 forms are poorly understood. However, evidence suggests that some may play an important role in the biosynthesis of plant cuticles. We review current knowledge on the induction and inhibition of activities as well as the regio- and stereo-specificity of the distinct forms so far characterised.

Characterisation of taurochenodeoxycholic acid 6 alpha-hydroxylase from pig liver microsomes.

A fraction of cytochrome P-450 catalysing an efficient 6 alpha-hydroxylation of taurine-conjugated 3 alpha,7 alpha-dihydroxy-5 beta- cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P-450 was 6 nmol/mg protein and the preparation showed two major protein bands upon SDS/PAGE. These two bands were isolated after SDS/PAGE and protein blotting. The protein band with a molecular mass of 53 kDa had an N-terminal amino acid sequence and internal sequences resembling that of the cytochrome P-450 4A subfamily (CYP 4A). Polyclonal antibodies raised against this protein were able to, after SDS/PAGE and immunoblotting, detect the protein in microsomal fractions as well as in the purified cytochrome P-450 fraction. Furthermore, addition of these antibodies to a reconstituted system containing the cytochrome P-450 fraction, inhibited 6 alpha-hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6 alpha-hydroxylation. The purified cytochrome P-450 fraction catalysed in addition omega- and omega-1 hydroxylation of lauric acid and 6 alpha-hydroxylation of 3 alpha-hydroxy-5 beta-cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6 beta-hydroxylation of taurochenodexycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5 beta-cholestane-3 alpha,7 alpha-diol. These results indicate that 6 alpha-hydroxylation of taurochenodeoxycholic acid is catalysed by a specific species of cytochrome P-450 that, according to N-terminal amino acid sequence as well as catalytic properties, could be a member of the CYP 4A subfamily.

Effects of perfluorooctanoic acid — a potent peroxisome proliferator in rat — on Morris hepatoma 7800C1 cells, a rat cell line

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 μM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and of cytochrome P-450IVA (specialized for omega-and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 μM PFOA has little effect on cellular morphology.

Valproate (VPA) metabolites in various clinical conditions of probable VPA-associated hepatotoxicity.

Of a cohort of 470 epileptic patients in whom valproate (VPA) serum metabolites had been measured, 170 subjects without symptoms or signs of hepatic side effects were chosen as a reference group to establish the usual metabolic pattern. A wide interindividual variation of VPA metabolite concentrations was noted. Infants receiving VPA monotherapy and comedication with other antiepileptic drugs (AEDs) showed lower concentrations of the potential hepatotoxin 4-ene-VPA than did older children. In 11 patients with early symptoms and signs of possible fatal VPA-associated hepatotoxicity, the following spectrum of benign clinical conditions was observed: unusually severe side effect during initiation of VPA therapy (1 patient), high VPA dosage (2 patients), reversible impairment of coagulation with bleeding manifestations in association with a slight increase in transaminase levels (1 child), and reversible liver dysfunction associated with febrile illness (7 patients). Reversible or irreversible fulminant liver failure had occurred in 5 children. Three of the 4 children with a fatal outcome had massive lactic acidosis. In all patients with probable VPA-associated hepatotoxicity, some aspects of VPA metabolism differed distinctly from that of the reference group, but the inter-individual profile of metabolites varied considerably, even in the subgroup of 4 children who died. Impairment of VPA beta-oxidation and increase of metabolites of alternative metabolic pathways (omega- and omega 1-hydroxylation, dehydrogenation reactions) were the most frequent findings. Increased values of 2-n-propyl-4-pentenoic acid metabolite of VPA (4-ene-VPA), could be detected only in 1 of the 5 patients with fulminant liver failure and in one other child with a slight hepatic dysfunction, indicating that this VPA metabolite is not the decisive hepatotoxin or indicator of hepatotoxicity. Because we cannot distinguish between benign and life-threatening hepatic adverse reactions on the basis of VPA metabolites, all identified changes are considered secondary to an as-yet-unknown primary metabolic event. The most toxic compound could be VPA itself, which may unmask an inborn or an acquired metabolic defect in the processing of fatty acids.

Perfluorooctanoic acid has persistent effects on peroxisome proliferation and related parameters in mouse liver.

Male C57Bl/6 mice were treated for 5 days with 0.05% perfluorooctanoic acid (PFOA) in their diet. This treatment resulted in a potent induction of peroxisomal fatty acid beta-oxidation in the liver. In order to investigate recovery from treatment with PFOA, mice were given normal laboratory chow for up to 20 days after termination of PFOA administration. It was established that the activities of peroxisomal lauoryl-CoA oxidase and palmitoyl-CoA oxidation were still elevated 2-3 weeks after termination of treatment. The catalase activity recovered in the cytosolic fraction was also still significantly elevated after 20 days with normal laboratory chow. Furthermore, the protein content of the mitochondrial fraction was increased by PFOA and had not returned to control level at the end of the recovery period. Perfluorooctanoic acid also caused a persistent effect in omega hydroxylation of lauric acid (cytochrome P-452). The activities of cytosolic DT-diaphorase and glutathione transferase were also enhanced by PFOA. However, these two enzymes recovered relatively rapidly from the treatment (2-20 days). This study reveals two different patterns of recovery from PFOA treatment, one involving parameters that recovered completely, or almost completely, from PFOA treatment after 20 days and another involving parameters that were still elevated at the end of the recovery period.

Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation of the terminal olefin by plant cytochrome P-450

The study of the stereochemistry and regioselectivity of plant fatty acid hydroxylases is hampered by the difficulty to purify plant cytochrome P-450 enzymes. To provide an alternative, we have now defined an experimental plant system which expresses only one hydroxylase activity towards lauric acid: microsomes from clofibrate-induced Vicia sativa seedlings hydroxylate this fatty acid exclusively at the methyl terminus. To explore the catalytic capabilities of this laurate oxidase, a series of 1- 14C-radiolabeled unsaturated lauric acid analogs (7-, 8-, 9-, 10- and 11-dodecenoic acids) were synthesized. Microsomes from clofibrate induced Vicia sativa seedlings catalyzed the omega-oxidation of the lauric acid analogs in the presence of O 2 and NADPH. The cis and trans forms of the four in-chain unsaturated analogs of lauric acid were 12-hydroxylated with similar efficiency. The terminal olefin was readily converted to the epoxide with only marginal autocatalytic inactivation of the enzyme. The formation of each metabolite was inhibited to the same extend when microsomes were incubated in presence of carbon monoxide or a suicide-substrate for omega LAH, suggesting that a single cytochrome P-450 isoenzyme from Vicia sativa microsomes is able to omega hydroxylate lauric acid and in-chain unsaturated analogs, and to epoxygenate 11-dodecenoic acid.

Cytochrome P450 and the arachidonate cascade.

Arachidonic acid and many products of the arachidonate cascade serve as substrates for cytochrome P450-mediated metabolism via allylic oxidation, omega hydroxylation, and epoxygenation as well as peroxide rearrangement. Defining the physiological importance of these metabolites is an area of intense research interest. Cytochrome P450-catalyzed reactions play prominent roles in multiplying the structural and functional diversity of the arachidonate metabolic cascade.

Stereoselective aliphatic hydroxylation of 6-n-propylchromone-2-carboxylic acid by female Dutch rabbits.

6-n-Alkylchromone-2-carboxylic acids are metabolized solely by aliphatic oxidation. In the rabbit, the 6-n-propyl congener (PCCA) undergoes omega-1 hydroxylation exclusively. Following administration of PCCA to female Dutch rabbits (500 mumol/kg), some 77% of the dose was excreted in the urine, 41% as PCCA and 36% as 6-(2'-hydroxy-n-propyl)chromone-2-carboxylic acid. Since this metabolite is chiral, we have examined the stereochemistry of the excreted material. Diastereoisomeric (as camphanate and alpha-methoxy-alpha-(trifluoro-methyl)phenylacetate esters) and direct chiral HPLC and chiral lanthanide shift NMR have each shown the S:R ratio of the excreted metabolite to be 76:24. When rabbits were dosed with the racemic metabolite, excretion of the compound was not stereoselective. The regio- and stereo-selectivity of the aliphatic hydroxylation of PCCA are thus reflections of the selectivities of the enzyme systems responsible for its formation and suggest PCCA to be an appropriate probe compound for the study of prochiral-chiral hydroxylations.

Mechanism-based probes of the topology and function of fatty acid hydroxylases.

The use of three mechanism-based probes to investigate the topology and function of fatty acid hydroxylases is discussed. 1) The observation of protein rather than heme alkylation in the reaction of cytochrome P4504A1 with 10-undecynoic acid supports the argument that the enzyme circumvents the inherent preference for omega-1 hydroxylation by restricting access to the ferryl oxygen. 2) The regiochemistry of the ferricyanide-mediated iron-to-nitrogen shift of the cytochrome P450102 (P450BM-3) phenyl-iron complex indicates that the active site of this bacterial fatty acid hydroxylase is open primarily above pyrrole ring A of the prosthetic heme group, 3) Inhibition of clofibrate-mediated peroxisome proliferation in cultured rat hepatocytes by inactivation of cytochrome P4504A1 indicates that omega-hydroxylation of fatty acids provides a signal for peroxisome proliferation.

Urinary organic acid excretion during feeding of medium-chain or long-chain triglyceride diets in patients with non-insulin-dependent diabetes mellitus

Medium-chain triglycerides (MCTs) are absorbed and metabolized differently from long-chain triglycerides (LCTs). Recent data indicate that MCTs may be useful as a dietary substitute in a variety of clinical disorders. The current studies were undertaken to characterize urinary organic acid excretion in patients with non-insulin-dependent diabetes mellitus during 4 d of an LCT or MCT diet. Urinary excretion of the dicarboxylic acids adipic, suberic, and 3-hydroxysebacic and the (omega-1) hydroxylation products 5-hydroxyhexanoic acid and 7-hydroxyoctanoic acid, was increased during MCT feeding as compared with LCT feeding. Urinary suberic and 7-hydroxyoctanoic acid excretions were increased 55- and 30-fold, respectively, during the MCT-substituted diet. Urinary organic acid profiles provide information on the fate of lipids during MCT feeding and may also be useful in assessing complicance during clinical trials employing MCT-substituted diets.