Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts

We demonstrate that naturally occurring C14 and C16-specific acylacyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA͞fadR mutant. Under the same growth conditions, C 18-specific ⌬ 9 -stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C 14 and C16 acyl-ACPs but a relative lack of C 18 acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor ⌬ 9 -18:0-ACP desaturase that display enhanced specificity for C 14 and C16 acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor ⌬ 9 -18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.A cyl-acyl carrier protein (ACP)-desaturases (DES) are a family of soluble enzymes that are associated with the synthesis of monounsaturated fatty acids in plants (1). These enzymes catalyze the insertion of a double bond into saturated fatty acids bound to ACP in the plastids of plant cells, and thus serve as one of the primary determinants of the unsaturated fatty acid content of plant membranes and seed storage oils. The most widely occurring member of this family is the ⌬ 9 -stearoyl (18:0 ‡ )-ACP DES, which introduces the double bond of oleic acid (1). In addition, a number of other acyl-ACP DES have been identified that are associated with the synthesis of unusual monounsaturated fatty acids and have altered substrate and regio-specifities relative to the ⌬ 9 -18:0-ACP DES. Variant acyl-ACP DES identified to date include the ⌬ 4 -palmitoyl (16:0)-ACP DES from Umbelliferae seed (2, 3), the ⌬ 6 -16:0-ACP DES from black-eyed Susan vine (Thunbergia alata) seed (4), the ⌬ 9 -myristoyl (14:0)-ACP DES from geranium (Pelargonium xhortorum) trichomes (5), and ⌬ 9 -16:0-ACP DES from cat's claw (Doxantha unguis-cati) (6) and milkweed (Asclepias syriaca) (7) seeds. These enzymes share Ն60% amino acid sequence identity with the ⌬ 9 -18:0-ACP DES.This collection of structurally related, but functionally diverged, enzymes provides a tool for understanding how acyl-ACP DES recognize the chain length of substrates and position the placement of double bonds. Characterization of these properties also has been greatly aided by information from the crystal structure of the castor ⌬ 9 -18:0-ACP DES (8). From the...

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“…The double-bond positions of monounsaturated fatty acid methyl esters were determined by GC-MS of dimethyl disulfide derivatives (21).…”

Section: Methodsmentioning

confidence: 99%

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Cahoon

Shanklin

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…The tracer:tracee ratio (TTR) was calculated for VA and RA from the mass abundance of the C. For VA, TTR was calculated from GC-MS single ion monitoring data on specific mass fragments at m/z 245 and 246 for M and M þ 1, respectively (25) , and using a standard curve of [ 13 C]trans-11-18 : 1. For RA, TTR was calculated from GC -MS scan data on specific mass fragments at m/z 312 and 313 for M and M þ 1, respectively (23) .…”

Section: Calculationsmentioning

confidence: 99%

Effects of fish oil and starch added to a diet containing sunflower-seed oil on dairy goat performance, milk fatty acid composition andin vivoΔ9-desaturation of [¹³C]vaccenic acid

et al. 2010

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The potential benefits on human health have prompted an interest in developing nutritional strategies for specifically increasing rumenic acid (RA) in ruminant milk. The aims of the present study were to (i) compare two dietary treatments with lipid supplements on milk yield and composition, (ii) measure the in vivo D9-desaturation of vaccenic acid (VA) to RA using 13 C-labelled VA and (iii) determine the effect of the dietary treatments on this variable. Treatments were 90 g sunflower-seed oil (SO) per d or 60 g sunflower-seed oil and 30 g fish oil per d plus additional starch (SFO), in a grassland hay-based diet given to eight Alpine goats in a 2 £ 2 cross-over design with 21 d experimental periods. Milk yield and composition were similar between treatments. Goats fed SFO had higher milk 6 : 0 -16 : 0 concentration, lower milk SC18 concentrations and showed no effect on milk VA and RA, compared with SO. At the end of the experiment, intravenous injection of 1·5 g [ 13 C]VA followed by measurements of milk lipid 13 C enrichment showed that in vivo 31·7 and 31·6 % of VA was D9-desaturated into milk RA in the caprine with the SO and SFO treatments, respectively. The expression of genes encoding for D9-desaturase (or stearoyl-CoA desaturase; SCD1, SCD5) in mammary tissues and four milk D9-desaturation ratios were similar between treatments. In conclusion, the present study provides the first estimates of in vivo endogenous synthesis of RA (63-73 % of milk RA) from VA in goats, and shows no difference between the two lipid supplements compared.

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“…Mass spectrometry was performed with an HP5973 mass selective detector (Hewlett-Packard). Double-bond positions of monounsaturated FAMEs were determined (25). Mass spectra, expected fragmentation patterns, and diagnostic ions for the molecular species identified are given in Fig.…”

Section: Materials Andmentioning

confidence: 99%

Switching desaturase enzyme specificity by alternate subcellular targeting

Heilmann

Pidkowich²,

Girke³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0⌬ 7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, ⌬ 7 to ⌬ 9 . Conversely, retargeting of two related cytoplasmic 16:0⌬ 9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity Ϸ25-fold, ⌬ 9 to ⌬ 7 . All three desaturases exhibited ⌬ 9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred ⌬ 7 desaturation, with 16:1⌬ 7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 ⌬ 7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic ⌬ 9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1⌬ 7 and its abundant metabolite, 16:3⌬ 7,10,13 . Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.M etabolic diversity in living systems arises primarily from biotransformations catalyzed by enzymes; indeed life itself depends on the specificity of enzymes. The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by the gradual accumulation of changes in the catalytic portion of the enzyme until new properties arise. However, there is an emerging body of evidence suggesting that an enzyme's functional characteristics can also be affected by its metabolic context and that temporal and spatial dynamics of enzyme interactions can be important determinants of enzyme functionality (1). Examples include the ''rewiring'' of mitogen-activated protein kinase pathways on alternative scaffolds (2); the localization-dependent interactions of transcription factors with RNA polymerase in the nucleus (reviewed in ref. 3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gati...

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Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts

Cited by 92 publications

References 34 publications

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Effects of fish oil and starch added to a diet containing sunflower-seed oil on dairy goat performance, milk fatty acid composition andin vivoΔ9-desaturation of [¹³C]vaccenic acid

Switching desaturase enzyme specificity by alternate subcellular targeting

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Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts

Cited by 92 publications

References 34 publications

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Effects of fish oil and starch added to a diet containing sunflower-seed oil on dairy goat performance, milk fatty acid composition andin vivoΔ9-desaturation of [13C]vaccenic acid

Switching desaturase enzyme specificity by alternate subcellular targeting

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Effects of fish oil and starch added to a diet containing sunflower-seed oil on dairy goat performance, milk fatty acid composition andin vivoΔ9-desaturation of [¹³C]vaccenic acid