The determination of double bond positions in polyunsaturated fatty acids–‐Gas chromatography/mass spectrometry of the diethylamide derivative

Nilsson, Ralf; Liljenberg, Conny

doi:10.1002/pca.2800020604

Cited by 36 publications

(21 citation statements)

References 14 publications

(12 reference statements)

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“…The fractions containing the fatty acids of interest were saponified (21) and derivatized to diethylamides according to Nilsson and Liljenberg (25). The diethylamide reaction products were further purified by applying the reaction mixture to a short silica gel column (ϳ 0.5 ml), washing with 10 column volumes of dichloromethane, and eluting the diethylamides with 10 column volumes of acetone.…”

Section: Table I Oligonucleotide Primers Used To Construct Chimeras Omentioning

confidence: 99%

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Sasata

Reed

Loewen

et al. 2004

Journal of Biological Chemistry

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Most fatty acid desaturases are members of a large superfamily of integral membrane, O 2 -dependent, ironcontaining enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans -3 (FAT-1) and "⌬12" (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as ؉3, i.e. it "measures" three carbons from an existing double bond. The structural basis for ؉3 and -3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from ؉3 to -3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region.Fatty acid desaturases are part of multicomponent systems that catalyze the oxygen-and nicotinamide adenine dinucleotidedependent syn-dehydrogenation of unactivated aliphatic regions of their fatty ester (acyl-lipid) or thioester (acyl-acyl carrier protein or acyl-CoA) substrates (1-6). In addition to the family of soluble fatty acid desaturases, of which the plant stearoyl-acyl carrier protein desaturase is well characterized, there is a large group of structurally distinct integral membrane desaturases. Membrane desaturases of widely varying substrate specificity and regioselectivity are scattered among a range of taxa. The yeast Saccharomyces cerevisiae has a single stearoyl-CoA ⌬9 desaturase, whereas many bacteria lack such desaturases entirely. At the other extreme, the biosynthesis of (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid in some marine fungi likely requires the successive action of six structurally similar membrane desaturases, each varying in substrate specificity and regioselectivity (7,8).Based on structural and catalytic similarities, membrane desaturases are included in a superfamily of oxidative enzymes along with alkane hydroxylase, xylene monooxygenase, carotene ketolase, and sterol methyloxidase (9, 10). These are thought to contain a histidine-coordinated diiron center at the active site. While essentially no three-dimensional structural information is available for these difficult to purify enzymes, primary structure similarity, especially the conservation of three histidine-rich motifs, and similarity o...

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Section: Table I Oligonucleotide Primers Used To Construct Chimeras Omentioning

confidence: 99%

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Sasata

Reed

Loewen

et al. 2004

Journal of Biological Chemistry

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“…For gas chromatography (GC) analysis, most of the fatty acids were transmethylated with methanolic/HCl (3 m) at 80°C for 1 to 2 h and then extracted with 2ϫ 2 mL of hexane. For acid-labile (conjugated) fatty acids and for double bond positional analysis, the fatty acids were derivatized with diethylamine according to the method of Nilsson and Liljenberg (1991). GC analysis was carried out as described by Taylor et al (1992).…”

Section: Growth and Biochemical Analysis Of Transformed Yeastmentioning

confidence: 99%

Characterization of the Brassica napusExtraplastidial Linoleate Desaturase by Expression in Saccharomyces cerevisiae

2000

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The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system. A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids. The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures. The results indicate that the enzyme has: (a) -3, as opposed to ⌬-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with -6 double bonds, but the ability to desaturate substrates with -6 hydroxyl groups or -9 or -5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate.

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“…; J & W Scientific) using helium as a carrier gas. Fatty acids were characterised by GC-MS after producing dimethyl disulphide adducts of monounsaturated fatty acids (Nichols et al 1986) and diethylamide derivatives of polyunsaturated fatty acids (Nilsson & Liljenberg 1991).…”

Section: Introductionmentioning

confidence: 99%

Lipid and lipid carbon stable isotope composition of the hydrothermal vent shrimp Mirocaris fortunata:evidence for nutritional dependence on photosynthetically fixed carbon

Pond¹,

Segonzac²,

Bell³

et al. 1997

Mar. Ecol. Prog. Ser.

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Mjrocaris fortunata were sampled from the Lucky Strike hydrothermal vent area (Eiffel Tower site) on the mid-Atlantic ridge during the French DlVA 2 cruise (June 1994). Small adults (17 to 22 mm total length), although morphologically identical, could be divided into 2 categones on the basis of pigmentat~on, llpid composition and I3C/l2c stable isotope ratios of fatty acids H~ghly pigmented small adults (8 6 to 9.2 pg carotenoid shrimp-') contamed higher levels of total lipid than similar-sized individuals containing lower levels of pigment (0.9 to 2 9 pg carotenoid shrimp-'). Lipid class analysis indicated that wax esters comprised 62.5% of total lipid in the former group. These p~gmented shrimp also contained high proportions of polyunsaturated fatty acids (PUFA), particularly the phototrophic microplanktonic markers 20:5(n-3) and 22:6(n-3) (14.0 and 33.5% respectively). By contrast small adults (22 mm) and adult shrimp (25 to 26 mm) with low levels of carotenoid pigmentation contained lower amounts of total lipid, little or no wax ester and low levels of 20:5(n-3) and 22:6(n-3), but did contain 16:2(n-4) and 18:2(n-4) and the non-methylene interrupted dienes 20:285,13 and 22:287,15. GC-IRMS analysis of all fatty acids and fatty alcohols in the pigmented small adults indicated 6I3C values of -18.2 to -27.7?&,, which is consistent with a photosynthetic carbon source for these compounds. The I3C/l2C Isotope composition of fatty acids from low-pigmented small adults and adults was more variable (-12 5 to -33.11:L) and suggests a bimodal distribution which may be attributable to differing nutritional sources or the physiological/reproductive status of these shnmp Samples of eggs, which are carried by the female on the pleopods, represented approximately 57 % of total somatic lipid which indicates a substantial reproductive investment by this species. The egg lipids comprised high proportions of triacylglycerols (64.4 to 78.0% of total I~pid) whilst the fatty acid composition was dominated by the monounsaturated fatty acids 16:l(n-71, 18:l(n-7) and 18:l(n-9). which accounted for 65.7 to 73.5"0 of total fatty acids. By contrast, PUFA were relatively minor components of egg lipids, particularly 20:5(n-3) and 22:6(n-3), which accounted for only 1.1 and 2.9% of total egg fatty acids respectively. This indicates that the reproductive investment by this species is supported mainly by material derived from bacterial chemosynthesis. The potential for M. fortunata hedge betting by producing larvae which either metamorphose at the vent site or adopt a bathypelagic lifestyle and delay metamorphosis to facilitate more widespread dispersal is discussed.

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The determination of double bond positions in polyunsaturated fatty acids–‐Gas chromatography/mass spectrometry of the diethylamide derivative

Cited by 36 publications

References 14 publications

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Characterization of the Brassica napusExtraplastidial Linoleate Desaturase by Expression in Saccharomyces cerevisiae

Lipid and lipid carbon stable isotope composition of the hydrothermal vent shrimp Mirocaris fortunata:evidence for nutritional dependence on photosynthetically fixed carbon

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