Microbial oxidation of oleic acid.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.     

Stereospecificity of Microbial Hydrations of Oleic Acid to 10-Hydroxystearic Acid

We recently described a simple method for ascertaining the stereochemical purities of hydroxy fatty acids (S. H. El-Sharkawy, W. Yang, L. Dostal, and J. P. N. Rosazza, Appl. Environ. Microbiol. 58:2116-2122, 1992) based on the ¹H-nuclear magnetic resonance spectral analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates. This report describes the stereochemistries of microbial hydrations of oleic acid to 10-hydroxystearic acid by Nocardia aurantia (also known as Rhodococcus rhodochrous) ATCC 12674, Nocardia restrictus ATCC 14887, Mycobacterium fortuitum UI-53387, Pseudomonas species strain NRRL-2994, Pseudomonas species strain NRRL B-3266, and baker's yeast. 10(R)-hydroxystearic acid isolated from Pseudomonas species strain NRRL-2994 was the standard for use in the ¹H-nuclear magnetic resonance spectral technique to permit simple assignments of the absolute configurations of 10-hydroxystearic acid produced by different microorganisms. While the R. rhodochrous ATCC 12674-mediated hydration of oleic acid gave mixtures of enantiomers 10(R)-hydroxystearic acid and 10(S)-hydroxystearic acid, Pseudomonas species strain NRRL-B-3266 produced optically pure 10(R)-hydroxystearic acid. The remaining microorganisms stereoselectively hydrated oleic acid to 10(R)-hydroxystearic acid containing between 2 and 18% of the contaminating 10(S)-hydroxystearic acid.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Inhibitors of sterol synthesis. Synthesis and spectral characterization of fatty acids esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

Reported herein are the chemical syntheses of 29 fatty acid esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These compounds were characterized by the results of ultraviolet, 1H nuclear magnetic resonance, and mass spectral studies.     

Microbial transformation of Ibuprofen by a nocardia species

The carboxylic acid functional group of ibuprofen [alpha-methyl-4-(2-methylpropyl) benzene acetic acid] is reduced to the corresponding alcohol and subsequently esterified to the acetate derivative by cultures of Nocardia species strain NRRL 5646. The alcohol and ester microbial transformation products were isolated, and their structures were determined by H and C nuclear magnetic resonance spectroscopy and mass spectrometry. By derivatization of synthetic and microbiologically produced ibuprofen alcohols with S(+)-O-acetylmandelic acid, nuclear magnetic resonance analysis indicated that the carboxylic acid reductase of Nocardia sp. is R enantioselective, giving alcohol products with an enantiomeric excess of 61.2%. The R enantioselectivity of the carboxylic acid reductase enzyme system was confirmed by using cell extracts together with ATP and NADPH in the reduction of isomeric ibuprofens.     

Biocatalytic Synthesis of Vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by ¹H nuclear magnetic resonance and mass spectral analyses.     

Glycosphingolipid headgroup orientation in fluid phospholipid/cholesterol membranes: similarity for a range of glycolipid fatty acids.

Galactosyl ceramide (GalCer) was labeled for nuclear magnetic resonance (NMR) spectroscopy by replacement of a hydrogen atom at C6 of the galactose residue with deuterium. Wideline 2H NMR of [d1]GalCer permitted consideration of a mechanism traditionally entertained for cell surface recognition site modulation: that the nature of the fatty acid attached to the sphingosine backbone of glycosphingolipids (GSLs) importantly influences carbohydrate headgroup orientation. Comparison was made among various glycolipid fatty acids by altering hydroxylation, saturation, and chain length. Studies were carried out in unsonicated bilayer membranes mimicking several important characteristics of cell plasma membranes: fluidity, low GSL content, predominant [sn-2]monounsaturated phosphatidylcholine (PC) (1-palmitoyl-2-oleoyl PC), and the presence of cholesterol. Spectroscopy was performed on samples over a range of temperatures, which included the physiological. 2H NMR spectra of [d1]GalCer having 18-carbon saturated fatty acid (stearic acid), cis-9-unsaturated fatty acid (oleic acid), D- and L-stereoisomers of alpha-OH stearic acid, or 24-carbon saturated fatty acid (lignoceric acid) were importantly similar. This argues that for GSLs dispersed as minor components in fluid membranes, variation of the glycolipid fatty acid does not provide as much potential for direct conformational modulation of the carbohydrate portion as has sometimes been assumed. However, there was some evidence of motional differences among the species studied. The 2H NMR spectra that were obtained proved to be more complex than was anticipated. Their features could be approximated by assuming a combination of axially symmetric and axially asymmetric glycolipid motions. Presuming the appropriateness of such a analysis, at a magnetic field of 3.54 T (23.215 MHz), the experimental spectra suggested predominantly asymmetric motional contributions. At the higher field of 11.7 T (76.7 MHz, equivalent to a proton frequency of 500 MHz), spectra indicated dominance by axially symmetric rotational modes. There was also evidence of some bilayer orientation in the stronger magnetic field. The unusual observation of spectral differences between the two magnetic field strengths may involve a diamagnetic response to high field on the part of some liposome physical characteristics.     

Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus

Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins.We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction.     

A revised structure of the antitumor drug elliptinium--amino(acid) adducts

Using simple organic reactions and spectrometric techniques (nuclear magnetic resonance and mass spectrometry), we propose a revised structure of the adducts obtained through the H2O2-peroxidase oxidation of the antitumor drug ellipticinium and aliphatic amino(acid) compounds. These derivatives display an oxazole ring structure between the O(9)- and N(10)- atoms.     

Differential effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells

The effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells were examined to identify fatty acid specific changes in lipid metabolism that might indicate a basis for the hypolipidemic effect attributed to eicosapentaenoic acid and related n-3 fatty acids. Cellular glycerolipid synthesis, as determined by [3H]glycerol incorporation, increased in a concentration-dependent manner in cells incubated 4 h with either eicosapentaenoic acid or oleic acid at concentrations between 10 and 300 microM. [3H]Glycerol-labeled triglyceride was the principal lipid formed and increased approximately fourfold with the addition of 300 microM oleic acid or eicosapentaenoic acid. Both fatty acids also produced a 20-40% increase in the total cellular triglyceride mass. Although both fatty acids increased triglyceride synthesis to similar extents, eicosapentaenoic acid-treated cells secreted 40% less [3H]glycerol-labeled triglyceride than cells fed oleic acid. Cellular synthesis of [3H]glycerol-labeled phosphatidylethanolamine and phosphatidylcholine was also reduced by 40% and 30%, respectively, in cells given eicosapentaenoic acid versus cells given oleic acid. Similar results were obtained in determinations of radiolabeled oleic acid and eicosapentaenoic acid incorporation. At a fatty acid concentration of 300 microM, incorporation of radiolabeled eicosapentaenoic acid into cellular triglycerides was greater than the incorporation obtained with radiolabeled oleic acid, while the reverse relationship was observed for the formation of phosphatidylcholine from the same fatty acids. Eicosapentaenoic acid is as potent as oleic acid in inducing triglyceride synthesis but eicosapentaenoic acid is a poorer substrate than oleic acid for phospholipid synthesis. The intracellular rise in de novo-synthesized triglyceride in eicosapentaenoic acid-treated cells without corresponding increases in triglyceride secretion suggests that eicosapentaenoic acid is less effective than oleic acid in promoting the transfer of de novo-synthesized triglyceride to nascent very low density lipoproteins.     

Biosynthesis and structural characterization of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas aeruginosa from fatty acids

In this study, we investigated the ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize poly(3-hydroxyalkanoates) (PHAs) from saturated fatty acids with an even number of carbon atoms, from eight to 22, and from oleic acid. In a non-limiting medium, all carbon sources but docosanoic acid supported cell growth and PHA production, with eicosanoic acid giving the highest yield. In magnesium-limiting conditions, higher yields were obtained from sources with up to 16 carbon atoms. Composition was estimated by gas chromatography of methanolyzed samples and (13)C nuclear magnetic resonance. The 3-hydroxyalkanoate units extended from hexanoate to tetradecanoate or tetradecenoate, with octanoate and decanoate as the predominant components. Weight average molecular weights ranged from 78,000 to 316,000. Fast atom bombardment mass spectrometry of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.     

Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina

NeuGc alpha 2-6Glc beta 1-1Cer (M5 ganglioside) and HSO3-8NeuGc alpha 2-6Glc beta 1-1Cer (T1 ganglioside) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a downfield-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.     

Structural determination of a new naturally occurring cyclic vitamin K

The respiratory quinone composition of 5 members of the genus Nocardia was examined. All species contained a hitherto unknown cyclic vitamin K-like molecule as their predominant lipoquinone. On the basis of mass spectrometry, 1H and 13C nuclear magnetic resonance spectrometry, the novel quinone was shown to correspond to (2E, 14E, 18E, 22E)-2-[3,7, 11,15,19,23-hexamethyl-25-(2,6,6-trimethylcyclohex-2- enyl)pentacosa-2, 14,18,22-tetraenyl]-3-methyl-1,4-naphthoquinone. Results indicate this molecule may represent a valuable phylogenetic marker for the prokaryote genus Nocardia.     

Compact oleic acid in HAMLET

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex between alpha-lactalbumin and oleic acid that induces apoptosis in tumor cells, but not in healthy cells. Heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of 13C-oleic acid in HAMLET, and to study the 15N-labeled protein. Nuclear Overhauser enhancement spectroscopy shows that the two ends of the fatty acid are in close proximity and close to the double bond, indicating that the oleic acid is bound to HAMLET in a compact conformation. The data further show that HAMLET is a partly unfolded/molten globule-like complex under physiological conditions.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Microbiological transformations of lipids: acyl-specific hydrolysis of lard by yeasts.

The fatty acid and positional specificities of Saccharomyces cerevisiae (UI-SACCH) and Schizosaccharomyces octosporus (NRRL Y-854) in the hydrolysis of lard were studied by using gas-liquid chromatography. Synthetic triglycerides were used to determine the positional specificities of the lipases of both organisms. Palmitic acid is specifically cleaved from all three triglyceride ester positions by S. cerevisiae, while S. octosporus was able to cleave stearic acid at either position 1 or position 3 of the glycerol moiety. Preparative scale fermentation with 200 g of lard per liter yielded 48.4 g of palmitic acid per liter with S. cerevisiae and 42 g of stearic acid per liter with S. octosporus. The free fatty acids produced by microbial transformation of lard were characterized spectrally (1H and C nuclear magnetic resonance and mass spectrometry) and chromatographically (thin-layer and gas chromatographies).     

H and 13C n.m.r. studies of serum from normal and Echinococcus multilocularis infected jirds

The major components of the 13C and high field region of the 1H nuclear magnetic resonance (n.m.r.) spectra of normal and Echinococcus multilocularis infected jirds were identified and compared. Substantial depletion of the glucose and fatty acid chains from lower density lipoproteins was detected in sera from infected animals. In addition, this proliferating metacestode markedly changed the appearance of the spectral region recently assigned to N-acetyl protons of carbohydrate side chains of N-acetylated glycoproteins.     

Novel fucolipids accumulating in human adenocarcinoma. I. Glycolipids with di- or trifucosylated type 2 chain

Among a series of fucolipids accumulating in human colonic and liver adenocarcinoma, two neutral fucolipids have been isolated and their carbohydrate structures have been characterized by methylation analysis, mass spectrometry, enzymatic degradation, and proton NMR spectrometry as shown in Structures 1 and 2 below. These glycolipids are either absent or present in very small quantity in normal colonic mucosa and normal liver tissue. (formular; see text) Difucosyl neolactonorhexaosylceramide (Structure 1) gives a doublet or a triplet on high performance thin layer chromatography and can be separated into four to five fractions on high pressure high performance liquid chromatography. These components have the same carbohydrate structure, but have different fatty acid composition. Fractions are characterized by a predominance of either alpha-hydroxy C16 fatty acid or alpha-hydroxy C24 fatty acid. Trifucosyl neolactonoroctaosylceramide (structure 2) gives two discrete bands, which are designated Z3 and Z4, on high performance thin layer chromatography. Methylation analysis and nuclear magnetic resonance spectra show that the Z3 and Z4 have identical carbohydrate structures. All the cases of cancer tissue that accumulate di- or trifucosyl structure (1 or 2 above) also accumulated lactofucopentaose(III)ceramide. A possible enhancement in a specific synthetic pathway including type 2 chain elongation coupled with alpha 1----3 fucosylation in human cancer is discussed.     

Comparison of interproton distances in DNA models with nuclear Overhauser enhancement data

The conformational flexibility inherent in the polynucleotide chain plays an important role in deciding its three-dimensonal structure and enables it to undergo structural transitions in order to fulfil all its functions. Following certain stereochemical guidelines, both right and left handed double-helical models have been built in our laboratory and they are in reasonably good agreement with the fibre patterns for various polymorphous forms of DNA. Recently, nuclear magnetic resonance spectroscopy has become an important technique for studying the solution conformation and polymorphism of nucleic acids. Several workers have used 1_H nuclear magnetic resonance nuclear Overhauser enhancement measurements to estimate the interproton distances for the various DNA oligomers and compared them with the-interproton distances for particular models of A and B form DNA. In some cases the solution conformation does not seem to fit either of these models. We have been studying various models for DNA with a view to exploring the full conformational space allowed for nucleic acid polymers. In this paper, the interproton distances calculated for the different stereochemically feasible models of DNA are presented and they are compared and correlated against those obtained from¹H nuclear magnetic resonance nuclear Overhauser enhancement measurements of various nucleic acid oligomers.     

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids

Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)