Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds.     

Absolute configuration of 3-hydroxy fatty acids present in lipopolysaccharides from various bacterial groups.

The absolute configuration of 3-hydroxy fatty acids has been studied, which are present in the lipopolysaccharides of the following bacteria: Phodopseudomonas gelatinosa, Rh. viridis, Rhodospirillum tenue, Chromobacterium violaceum, Pseudomonas aeruginosa, Bordetella pertussis, Vibrio metchnikovii, Vibrio cholerae, Salmonella spp., Escherichia coli, Shigella flexneri, Proteus mirabilis, Yersinia enterocolitica and Fusobacterium nucleatum. The 3-hydroxy acids were liberated by strong alkaline hydrolysis, converted to 3-methoxy acid L-phenylethylamides and analyzed by gas-liquid chromatography. With the aid of authentic D-3-hydroxy fatty acids it was shown for all lipopolysaccharides that the 3-hydroxy acids, regardless of chain lengths, branching, 3-O-substitution or type of linkage, possess the D-configuration. 2-Hydroxydodecanoic acid, which is present in some lipopolysaccharides, was analyzed in an analogous way and shown to possess the L-configuration.     

A simple method for the extraction and quantification of photopigments from Symbiodinium spp.

We have developed a simple, mild extraction procedure using methanol which, when coupled with HPLC analysis and diode array detection (DAD), can be used to quantify the major photopigments found in cultured Symbiodinium spp. Extracts were prepared by suspending, fresh or frozen (− 70 °C), wet cell pellets in methanol and sonicating or not sonicating the cell suspensions before soaking the cells for 2 h in an ice bath. To assist the soaking process, cell suspensions were vortex mixed at 30 min intervals. After soaking, 0.5 M ammonium acetate buffer was added (1 part buffer to 9 parts methanol) before suspensions were stored over night at − 20 °C. Greater than 92% the recoverable pigment was obtained in the initial extraction of the four major photopigments, chlorophyll c, peridinin, diadinoxanthin, and chlorophyll a. Neither sonication nor freezing substantially increased the recovery of photopigments extracted with methanol. Extraction by other commonly used solvents such as acetone or acetone:water with or without freezing and sonication were less effective.     

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

Differential regulation of dihydroceramide desaturase by palmitate versus monounsaturated fatty acids: implications for insulin resistance

Much data implicate saturated fatty acids in deleterious processes associated with obesity, diabetes, and the metabolic syndrome. Many of these changes may be due to aberrant generation of bioactive lipids when saturated fatty acid availability to tissues is increased. On the other hand, studies are emerging that implicate the monounsaturated fatty acid oleate in protection from saturated fat mediated toxicity; however, the mechanisms are not well understood. Our data demonstrate a novel role for palmitate in increasing mRNA encoding DES1, which is the enzyme responsible for generating ceramide from its precursor dihydroceramide and thus controls synthesis of the bioactive lipid ceramide. Moreover, co-treatment with oleate prevented the increase in ceramide, and this occurred through attenuation of the increase in message and activity of DES1. Knockdown of DES1 also protected from palmitate-induced insulin resistance, and overexpression of this enzyme ameliorated the protective effect of oleate. Together, these findings provide insight into the mechanisms of oleate-mediated protection against metabolic disease and provide novel evidence for fatty acid-mediated regulation of a key enzyme of ceramide biosynthesis.     

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.     

A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles

A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%.     

Picolinyl esters for the structural determination of fatty acids by GC/MS

The position of unsaturation, chain branching, and other structural features of fatty acids are not often apparent from the mass spectra of common derivatives such as methyl esters because of factors such as charge location at the carboxy termiunus and migration of double bonds. The spectra of picolinyl esters, on the other hand, contain fragment ions that provide this information. The esters are synthesized by reaction of the acids with thionyl chloride to form the acid chloride that is reacted with 3-pyridylcarbinol to give the ester. Under electron impact conditions in the mass spectrometer, an electron is removed from the nitrogen of the pyridine ring and a hydrogen atom is abstracted from the alkyl chain to this electron-deficient site. This process produces a radical site in the chain that initiates chain cleavage. Hydrogen atoms can be removed from any position of the chain with varying probability, depending on the chain structure. Thus, diagnostic ions are produced from each type of fatty acid whose masses and relative abundances reflect the structure of the alkyl chain and any substituents. Patterns of fragmentation for straight-chain, branched-chain, unsaturated and cyclic fatty acids are described together with those containing hydroxy-, epoxy-, keto-, and ether groups.     

Effect of unsaturation in fatty acids on the binding and oxidation by myeloperoxidase: Ramifications for the initiation of atherosclerosis

Oxidation of low density lipoproteins (LDL) in the presence of myeloperoxidase and subsequent uptake of the oxidized LDL by specialized receptors on macrophages has been suggested as an initiating event of atherosclerosis. Oxidized fatty acid chains within the glycerophospholipids of LDL have been implicated as the recognition feature by the receptors. The ability of three fatty acids (oleic, linoleic, and arachidonic acids) typically contained in the lipid portion of the glycerophospholipids to bind and be oxidized by myeloperoxidase was measured by spectroscopically observing interactions of the lipids with the heme prosthetic group of the enzyme. As unsaturation increases in the lipid chain, myeloperoxidase binds and oxidizes the fatty acid more readily, as measured by K_D, K_M, and k_cat. A possible mechanism of the free radical oxidation by myeloperoxidase is discussed.     

Use of phages to control Campylobacter spp.

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.     

Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis

Stearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) protein that catalyzes the Delta9-cis desaturation of saturated fatty acids. Mice with targeted disruption in SCD1 (Scd1(-/-)) have significant reduction in the tissue content of triglycerides, suggesting that monounsaturated fatty acids endogenously synthesized by SCD1 are important for triglyceride synthesis. Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is the enzyme that catalyzes the final reaction in the synthesis of triglycerides. The lack of DGAT2, one of the two DGAT isoforms, results in almost a complete loss of tissue triglycerides. We hypothesize that SCD1 participates in triglyceride synthesis by providing a more accessible pool of monounsaturated fatty acids through substrate channeling. In this study, we test whether SCD1 is proximal to DGAT2 by colocalization study with confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer using HeLa cells as the model of study. All of the results suggest that SCD1 and DGAT2 are located very close to each other in the ER, which is a very important criterion for the channeling of substrate. By performing subcellular fractionation using mouse livers, we also show, for the first time, that SCD is present in the mitochondria-associated membrane.     

Sterols and fatty acids of the marine diatom biddulphia sinensis

Nine sterols, most showing Δ⁵- or Δ^5,22-unsaturation, were identified in the marine diatom Biddulphia sinensis. One sterol, cholesta-5,22E-dien-3β-ol, comprised 70–80% of the total sterols which is the first such predominance noted in a diatom. The only Δ⁷-sterol detected was cholest-7-en-3β-ol and this was a very minor component. A sterol showing unusual side-chain alkylation,23,24-dimethylcholesta-5,22E-dien-3β-ol, was identified for the first time in a diatom. Total fatty acids exhibited a predominance of Δ⁹- 16:1, 14:0, 20:5 and 16:0, typical of diatoms, although the proportions of these acids were found to vary with culture maturity. n-Heneicosahexaene was the major hydrocarbon together with a small amount of squalene.     

Signature fatty acids in the polar lipids of acid-producing Thiobacillus spp.: Methoxy, cyclopropyl, alpha-hydroxy-cyclopropyl and branched and normal monoenoic fatty acids

Abstract The polar lipids of 5 species of Thiobacillus were extracted and purified. An analysis of the fatty acid composition of the polar lipids documented the presence of methoxy, cyclopropyl, monounsaturated and hydroxycyclopropyl fatty acids of sufficiently unusual structure to serve as 'signatures' for the presence of these organisms in environmental samples. The structures of the unusual fatty acids of the polar lipids were confirmed by mass spectrometry (MS) after isolation by capillary gas chromatography (GC).     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Polyunsaturated fatty acids down-regulate in vitro expression of the key intestinal cholesterol absorption protein NPC1L1: no effect of monounsaturated nor saturated fatty acids

Several transporter proteins regulate intestinal cholesterol absorption. Of these proteins, NPC1L1 is a major contributor to this process. Fatty acids (FAs) modulate cholesterol absorption by a mechanism that remains unknown. We evaluate the effect of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) on the expression of NPC1L1 and others proteins associated with cholesterol absorption (SR-BI, ABCG5, ABCG8, ABCA1, CAV-1, ANX-2) in human enterocytes in vitro. The role of SREBPs, PPARs, LXR and RXR in this process was also investigated. Caco-2/TC-7 enterocytes were incubated for 24 h with a wide range of concentrations of FA–bovine serum albumin (50–300 μM). Gene expression was analyzed by quantitative real-time PCR. The NPC1L1 protein present in enterocyte membranes was analyzed using Western blot. NPC1L1 mRNA levels were reduced 35–58% by the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P<.05). Linoleic acid (n-6), palmitic acid and oleic acid did not affect NPC1L1 mRNA expression. ABCA1 mRNA levels were reduced 44–70% by n-6 arachidonic acid and 43–55% by n-3 EPA (P<.05). LXR and LXR+RXR agonists decreased NPC1L1 mRNA expression by 28% and 57%, respectively (P<.05). A concentration of 200 μM of EPA and DHA decreased NPC1L1 protein expression in enterocyte membranes by 58% and 59%, respectively. We have demonstrated that the PUFAs n-3 EPA and DHA down-regulate NPC1L1 mRNA expression. In addition, PUFAs also down-regulate NPC1L1 protein expression in enterocyte membranes. LXR and RXR activation induced a similar repression effect. The lipid-lowering effect of n-3 PUFAs could be mediated in part by their action at the NPC1L1 gene level.     

Very-long-chain iso and anteiso branched fatty acids in N-acylphosphatidylethanolamines from a natural cyanobacterial mat of Calothrix sp.

A combination of TLC, ESI-MS/MS and GC-MS was used to identify unusual molecular species of N-acylphosphatidylethanolamines containing very-long-chain anteiso branched fatty acids (VLCFAs) from Calothrix sp. collected in Antarctica and determine their component VLCFA up to 33-methyltetratriacontanoic acid as picolinyl ester derivatives using GC-MS.