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.     

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.     

Clofibrate-inducible rat hepatic P450s IVA1 and IVA3 catalyze the omega- and (omega-1)-hydroxylation of fatty acids and the omega-hydroxylation of prostaglandins E1 and F2 alpha

Cytochromes P450IVA1 and IVA3 display 72% amino acid sequence similarity and are expressed in livers of rats treated with the hypolipidemic drug clofibrate. The catalytic activities of IVA1 and IVA3 were examined by cDNA-directed expression using vaccinia virus. cDNA-expressed IVA1 and IVA3 had relative Mrs of 51,500 and 52,000, respectively, on SDS-polyacrylamide gels. Both enzymes displayed reduced, CO-bound absorption spectra with lambda max of 452.5 nm. IVA1 and IVA3 hydroxylated lauric acid at the omega and omega-1 positions with equivalent omega/omega-1 ratios of about 12.5. IVA1 had a substrate turnover of 21 min-1 which was about fourfold higher than that of IVA3. The omega and omega-1 hydroxylation of palmitic acid was also catalyzed by these P450s with combined turnover numbers for both metabolites of 45 min-1 or 18 min-1 for IVA1 and IVA3, respectively. The omega/omega-1 oxidation ratio of IVA1 for palmitate was 1.25 which was almost fourfold higher than that obtained for IVA3. These enzymes also catalyzed omega oxidation of the physiologically important eicosanoids prostaglandins E1 and F2 alpha with turnover numbers of about one-tenth those calculated for fatty acid oxidations. No omega-1 hydroxy metabolites were produced. These studies indicate that the P450 enzymes IVA1 and IVA3 are able to catalyze the oxidations of both fatty acids and prostaglandins.     

Fatty acid omega and (omega-1)-Hydroxylation in rabbit intestinal mucosa microsomes.

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid.Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.     

The numerous effector functions of Nef.

P450BM-3, a catalytically self-sufficient, soluble bacterial P450, contains on the same polypeptide a heme domain and a reductase domain. P450BM-3 catalyzes the oxidation of short- and long-chain, saturated and unsaturated fatty acids. The three-dimensional structure of the heme domain both in the absence and in the presence of fatty acid substrates has been determined; however, the fatty acid in the substrate-bound form is not adequately close to the heme iron to permit a prediction regarding the stereoselectivity of oxidation. In the case of long-chain fatty acids, the products can also serve as substrate and be metabolized several times. In the current study, we have determined the absolute configuration of the three primary products of palmitic acid hydroxylation (15-, 14-, and 13-OH palmitic acid). While the 15- and 14-hydroxy compounds are produced in a highly stereoselective manner (98% R, 2% S), the 13-hydroxy is a mixture of 72% R and 28% S. We have also examined the binding of these three hydroxy acids to P450BM-3 and shown that only two of them (14-OH and 13-OH palmitic acid) can bind to and be further metabolized by P450BM-3. The results indicate that in contrast to the flexibility of palmitoleic acid bound to the oxidized enzyme, palmitic acid is rigidly bound in the active site during catalytic turnover.     

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of omega-1, omega-2 and omega-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.     

Fusarium oxysporum fatty-acid subterminal hydroxylase (CYP505) is a membrane-bound eukaryotic counterpart of Bacillus megaterium cytochrome P450BM3

The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast. The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium. The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3. Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells. This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane. These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems. P450foxy was classified in the new family, CYP505.     

Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum

The cell-free extract of a cytochrome P-450-producing fungus, Fusarium oxysporum, was found to catalyze the hydroxylation of fatty acids. Three product isomers were formed from a single fatty acid. The products from lauric acid were identified by mass-spectrometry as 9-, 10-, and 11-hydroxydodecanoic acids, and those from palmitic acid as 13-, 14-, and 15-hydroxyhexadecanoic acids. The ratio of the isomers formed was 50 : 36 : 14 in the case of laurate hydroxylation, and 37 : 47 : 16 in the case of palmitate. The reaction was dependent on both NADPH (or NADH) and molecular oxygen,and was strongly inhibited by carbon monoxide, menadione, or the antibody to purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450 with an apparent Kd of 0.3 mM. The hydroxylase activity together with cytochrome P-450 could be detected in both the soluble and microsome fractions, and the activity was almost proportional to the amount of cytochrome P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 is involved in the (omega-1)-, (omega-2)-, and (omega-3)-hydroxylation of fatty acids catalyzed by the cell-free extract of the fungus.     

Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Mechanisms involved in the differential reduction of omega-3 and omega-6 highly unsaturated fatty acids by structural heart disease resulting in "HUFA deficiency"

The causes of reduced levels of omega-3 and omega-6 highly unsaturated fatty acids ("HUFA deficiency") in heart failure remain unresolved. HUFA profiles were examined in the serum of 331 patients with failing versus nonfailing heart disease. Arachidonic acid was positively correlated (P?< 0.001) with eicosapentaenoic acid (EPA) (r = 0.40) and docosahexaenoic acid (DHA) (r = 0.53) and negatively with palmitic (r = 0.42), palmitoleic (r = 0.38), and oleic acid (r = 0.48). Delta-5 desaturase activity was reduced (P?< 0.01) in heart failure patients with low ejection fraction, dilatation, increased wall stress, and reduced heart rate variability (SDNN). In these patients, the reduced (P?< 0.01) HUFA and increased palmitic (P?< 0.01) and oleic acid (P = 0.05) arose from separate influences involving reduced cardiac contractility (arachidonic acid and palmitic acid predicted by ejection fraction) and chamber dilatation (DHA and oleic acid predicted by end-diastolic diameter). A low DHA (0.2%-0.9% versus 1.4%-3.1%) was associated (P?< 0.025) with atrial dilatation (44?± 8?mm versus 40?± 8?mm). Equidirectional but less pronounced effects on HUFA were induced by sympathetic activation and (or) insulin resistance (fat and sugar fed to deoxycorticosterone acetate (DOCA)-salt rats) but not by compensated cardiac overload alone (DOCA-salt or aortic constriction), or reduced fatty acid oxidation (CPT-1 inhibition). Based on administration of omega-3 HUFA (OMACOR), dilatation is identified as a target for 1-2?g omega-3 HUFA·day(-1). Interventions for reduced arachidonic acid remain to be explored.     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Oxygenation of omega-3 fatty acids by human cytochrome P450 4F3B: effect on 20-hydroxyeicosatetraenoic acid production

Cytochrome P450 (CYP) omega-oxidases convert arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), a lipid mediator that modulates vascular tone. We observed that a microsomal preparation containing recombinant human CYP4F3B, which converts AA to 20-HETE, converted eicosapentaenoic acid (EPA) to 20-OH-EPA. Likewise, docosahexaenoic acid (DHA) was converted to 22-OH-DHA, indicating that human CYP4F3B also can oxidize 22-carbon omega-3 fatty acids. Consistent with these findings, addition of 0.5-5 microM EPA, DHA or omega-3 docosapentaenoic acid (DPA) to incubations containing 0.5 microM [3H]AA inhibited [3H]20-HETE production by 15-65%. [3H]20-OH-EPA was rapidly taken up by COS-7 cells, and almost all of the incorporated radioactivity remained as unmodified 20-OH-EPA. The 20-OH-EPA stimulated luciferase activity in COS-7 cells that express peroxisome proliferator-activated receptor alpha, indicating that this EPA metabolite may function as a lipid mediator. These findings suggest that some functional effects of omega-3 fatty acid supplementation may be due to inhibition of 20-HETE formation or the conversion of EPA to the corresponding omega-oxidized product.     

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.     

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.     

Substrate specificity of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis> in the hydroxylation of unsaturated fatty acid

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol).     

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.     

Requirement for omega and (omega;-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega;-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega;-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega;-1)/omega ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega;-1)-hydroxylation inside the access channel of active site.     

Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients

Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.