Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ⁶-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ⁹-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ⁶ or Δ⁹ positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ⁶-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ⁹-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ⁹-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ⁹-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.     

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

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(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 C(16) 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 delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) 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 delta(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 single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.     

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Seed oils are characterized by diverse fatty acid (FA) structures (1–3). Many of these deviate from typical C16 (e.g., palmitic acid, 16:0) and C18 (e.g., oleic acid, 18:1Δ9cis) FA that occur widely in seed oils, including major commercial vegetable oils. Structurally variant FAs, often referred to as “unusual” FAs, can have chain lengths other than 16 or 18 carbon atoms, double bonds in nontypical positions or cis-trans configurations, or carbon-chain modifications, such as hydroxyl or epoxy groups (4). The study of unusual FA biochemistry has increased understanding of enzyme structural determinants of substrate binding and reaction outcomes (4). These studies have also revealed how variations in biosynthetic and metabolic pathways allow for high levels of unusual FA biosynthesis and accumulation and have also facilitated research on the evolution of biochemical diversity in plants. Unusual FAs can occur in selected species or arise throughout a genus or family, and their accumulation may be associated with compensatory mutations in enzymes beyond the initial biosynthetic enzyme (5). Moreover, genes for unusual FA biosynthesis and metabolism can be useful for biotechnological research in plants and microbes to develop vegetable oils with enhanced nutritional or industrial value (6).In a variety of taxa, metabolic variations result in “extreme” unusual FA accumulation to ≥90% of seed oil, which can account for ≤50% of seed weight (2). Examples of extreme unusual FA production include medium chain-length FAs (C8–C14) in seeds of Cuphea species and ricinoleic acid, a hydroxylated C18 monounsaturated FA in castor seeds (Ricinus communis) (7, 8). Studies of FA metabolism in Cuphea have uncovered structurally variant FatB acyl-acyl carrier protein (ACP) thioesterases and acyltransferases associated with the biosynthesis and accumulation of medium chain-length FAs. These variant thioesterases have provided primary sequences for uncovering information about amino acid residues that control their substrate recognition properties (9–11). Studies of FA metabolism in castor seeds led to the discovery that structural variations in FAD2 genes for Δ12-oleic acid desaturases can lead to variant FA modifications, including insertion of hydroxyl and epoxy groups, triple bonds, and conjugated double bonds (12–14). FA biosynthetic and metabolic genes associated with these pathways have also been used to develop new oil functionalities in existing oilseed crops.Studies of unusual monounsaturated FAs have provided a wealth of biochemical information and biotechnological utility (15). Oleic acid, the most widely occurring monounsaturated FA in plants, is synthesized by the soluble Δ9-stearoyl-ACP desaturase (16). Structural variants of this enzyme result in a number of unusual FAs, including palmitoleic acid (16:1Δ9), sapienic acid (16:1Δ6), and petroselinic acid (18:1Δ6) (15). Studies of variant acyl-ACP desaturases in combination with three-dimensional (3D) structural data (17, 18) have revealed amino acid residues that control FA chain length- and regio-specificities of this enzyme class (19). This information has facilitated biotechnological development of oilseeds with high levels of unusual monounsaturated FAs using rationally designed acyl-ACP desaturases (20). In the case of petroselinic acid, a biosynthetic pathway has been deduced that involves not only a variant Δ4-16:0-ACP desaturase but also a coevolved variant β-ketoacyl-ACP synthase I and acyl-ACP thioesterase that together generate petroselinic acid at ≤85% of the total FAs of Apiaceae and Araliaceae seeds (21–24). This highlights that unusual FA biosynthesis can result from the initial evolution of a specialized biosynthetic enzyme, followed by evolution of additional enzymes that facilitate unusual FA production and accumulation.As part of our larger efforts to characterize the evolution of unusual FA biosynthesis in plants, we examined the FA composition of seeds in diverse Thunbergia species. It has been previously shown that Thunbergia alata seeds accumulate sapienic acid (16:1Δ6cis) to ∼85% of total FAs via the activity of a Δ6-16:0-ACP desaturase (25, 26). As reported here, we discovered that seeds of several Thunbergia species accumulate petroselinic acid instead of sapienic acid. In these seeds, petroselinic acid accumulates >90% of total FAs, which is among the highest naturally occurring levels of monounsaturated FA in plant seed oils. We provide biochemical, structural, and genetic evidence to explain the divergent evolution of increased sapienic and petroselinic acid production in Thunbergia. Furthermore, we show that the petroselinic acid biosynthetic pathway in Thunbergia is distinct from that in Apiaceae and Araliaceae (21) and can guide biotechnological production of unusual monounsaturated FAs in engineered crops and microbes.     

Fatty acid desaturases in human adipose tissue: relationships between gene expression, desaturation indexes and insulin resistance

Aims/hypothesis Fatty acid desaturases introduce double bonds into growing fatty acid chains. The key desaturases in humans are Δ⁵-desaturase (D5D), Δ⁶-desaturase (D6D) and stearoyl-CoA desaturase (SCD). Animal and human data implicate hepatic desaturase activities in insulin resistance, obesity and dyslipidaemia. However, the role of desaturase activity in adipose tissue is uncertain. We therefore evaluated relationships between adipose mRNA expression, estimated desaturase activities (fatty acid ratios) in adipose tissue and insulin resistance. Methods Subcutaneous adipose tissue mRNA expression of D5D (also known as FADS1), D6D (also known as FADS2) and SCD was determined in 75 individuals representative of the study population of 294 healthy 63-year-old men. Desaturation indexes (product/substrate fatty acid ratios) were generated from adipose tissue fatty acid composition in all individuals. Insulin resistance was defined as the upper quartile of the updated homeostasis model assessment (HOMA-2) index. Results The relevant desaturation indexes (16:1/16:0, 18:1/18:0, 20:4/20:3 and 18:3/18:2) reflected expression of SCD, but not of D5D or D6D in adipose tissue. Insulin-resistant individuals had a higher adipose tissue 18:1/18:0, but not 16:1/16:0 ratio than insulin-sensitive individuals. Individuals with a high adipose tissue 18:1/18:0 ratio were 4.4-fold (95% CI 1.8–11.8) more likely to be insulin resistant [threefold (95% CI 1.1–8.6) after adjustment for waist circumference and plasma triacylglycerol]. In a multiple regression model predicting HOMA-2, the independent effect of the 18:1/18:0 ratio was borderline (p = 0.086). Conclusions/interpretation Adipose tissue desaturation indexes of SCD reflect the expression of the gene encoding the enzyme in this tissue. Elevated SCD activity within adipose tissue is closely coupled to the development of insulin resistance.     

Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ^6,9,12) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a Δ⁶-fatty acid desaturase using linoleic acid (C18:2 Δ^9,12) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ⁶-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 Δ^6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ⁶-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b₅.     

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.     

Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

Pulse-chase experiments with [³H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a ³H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi [³H]acylprotein and [³H]palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (“34K”) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. Phosphate ions and, to a lesser extent, organic alcohols stimulated the rate of acyl-protein cleavage. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.     

Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants

We report the identification of bifunctional Delta12/omega3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea. The bifunctional activity of these desaturases distinguishes them from all known Delta12 or omega3 fatty acid desaturases. The omega3 desaturase activity of these enzymes also shows a broad omega6 fatty acid substrate specificity by their ability to convert linoleic acid (LA), gamma-linolenic acid, di-homo-gamma-linolenic acid, and arachidonic acid to the omega3 fatty acids, alpha-linolenic acid (ALA), stearidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid (EPA), respectively. Phylogenetic analysis suggests that omega3 desaturases arose by independent gene duplication events from a Delta12 desaturase ancestor. Expression of F. moniliforme Delta12/omega3 desaturase resulted in high ALA content in both Yarrowia lipolytica, an oleaginous yeast naturally deficient in omega3 desaturation, and soybean. In soybean, seed-specific expression resulted in 70.9 weight percent of total fatty acid (%TFA) ALA in a transformed seed compared with 10.9%TFA in a null segregant seed and 53.2%TFA in the current best source of ALA, linseed oil. The ALA/LA ratio in transformed seed was 22.3, a 110- and 7-fold improvement over the null segregant seed and linseed oil, respectively. Thus, these desaturases have potential for producing nutritionally desirable omega3 long-chain polyunsaturated fatty acids, such as EPA, with a significantly improved ratio of omega3/omega6 long-chain polyunsaturated fatty acids in both oilseeds and oleaginous microbes.     

A vertebrate fatty acid desaturase with Δ5 and Δ6 activities

Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity. The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.     

Functional expression of a Delta12 fatty acid desaturase gene from spinach in transgenic pigs

Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximately 10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximately 20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.     

Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.Sex pheromones (SPs) are a diverse group of chemical compounds that are central to mate-finding behavior in insects (1). Variation in SP composition between closely related species and among populations is well documented. Despite this variation, SPs are presumed to be under strong stabilizing selection, and thus the genetic mechanisms driving SP diversification represented an enigma (2). Research on SPs in moths (Insecta: Lepidoptera) helped establish the hypothesis of asymmetric tracking as a major driving force in SP diversification. In this scenario, abrupt changes in female SP composition via a shift in component ratio or the inclusion or loss of a component result in a distinct SP that attracts males with more broadly or differentially tuned SP preference (3). Assortative mating, the preferential mating of females producing a novel SP with males attracted to this SP, restricts gene flow between subpopulations with differing SP compositions. This can ultimately lead to speciation and fixation of novel communication channels (4). Work in insect models such as wasps (5), fruit flies (6), and especially moths (7–9) is helping uncover the genetic basis of SP diversification.In the majority of moth species, females use species-specific mixtures of SP components (SPCs) consisting of volatile fatty acid (FA) derivatives to attract conspecific males at long range. These SPCs are predominantly long-chain aliphatic (C12–C18) acetates, alcohols, or aldehydes containing zero to three double bonds of various configurations at different positions along the carbon backbone (10). Pheromone biosynthesis involves modifications of fatty acyl substrates, such as chain shortening and elongation, reduction, acetylation, oxidation, and desaturation (11). SP biosynthetic enzymes [i.e., FA reductases (8), FA chain-shortening enzymes (12, 13), and particularly FA desaturases (FADs) (7, 9, 14–17)] are the most commonly discovered traits underlying SP divergence in moths.Manduca sexta females attract males by releasing an SP containing in addition to mono- and diunsaturated aldehydes, which are typical structural themes in SPs of Bombycoidea moths (10), also uncommon conjugated triunsaturated aldehydes. The production of triunsaturated SPCs represents an easily traceable phenotype, thus making M. sexta a convenient yet unexploited model organism for unraveling the mechanisms of chemical communication evolution via novel SPC recruitment. In our previous attempts to decipher the desaturation pathway leading to triunsaturated SPC FA precursors (3UFAs), we identified the MsexD2 desaturase, which exhibits Z11-desaturase and conjugase (1,4-dehydrogenase) activity and participates in stepwise production of monounsaturated (1UFA) and diunsaturated (2UFA) SPC precursors. The terminal desaturation step resulting in the third conjugated double bond remained, however, elusive (18, 19).Here, we isolated and functionally characterized FAD genes abundantly and specifically expressed in the pheromone gland (PG) capable of producing 3UFA pheromone precursors and demonstrated the biosynthesis of 3UFAs from 2UFAs. We used site-directed mutagenesis of M. sexta FADs and identified a minimal structure motif leading to acquisition of new desaturase specificities. The reconstructed evolutionary relationship of moth FADs demonstrated that the 3UFA pheromone precursors in M. sexta were acquired via (i) activation of a presumably inactive ancestral FAD gene and/or (ii) duplication of an ancestral FAD gene producing 1UFA and 2UFA SPC precursors followed by functional diversification of an FAD duplicate.     

The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation

The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7′E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7′E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate.     

Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids

Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, that makes E11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene precursor is made available by beta oxidation of E11-16 acid with a subsequent two-carbon loss to E9-14 acid. A functional assay using a baculovirus expression system required addition of myristic acid and E9-14 acid precursors to demonstrate the unusual regiospecificity and stereospecificity of this desaturase. The amino acid sequence of this desaturase has approximately 61% identity to that of Z11-desaturases from two other insect species, and only approximately 48% identity to the metabolic Z9-desaturases in those species. A pheromone-gland Z9-desaturase gene also was found with the light brown apple moth that differed in its deduced amino acid sequence (66% identity) with the metabolic Z9-desaturase from fat body in this species.     

Fatty acid composition of serum lipids predicts the development of the metabolic syndrome in men

Aims/hypothesis Types of dietary fat have been related to components of the metabolic syndrome. Serum fatty acid composition mainly reflects dietary fat intake, but also endogenous fatty acid synthesis catalysed by -desaturases. It is not known whether alterations of fatty acid composition or desaturase activities predict metabolic syndrome.Materials and methods We prospectively evaluated fatty acid composition in serum cholesteryl esters and estimated desaturase activities in 1,558 50-year-old men taking part in a population-based cohort study. The follow-up time was 20 years. Stearoyl-CoA desaturase (SCD-1), 6 (D6D) and 5 (D5D) desaturases were estimated as precursor to fatty acid ratios.Results High activity of estimated SCD-1 (odds ratio=1.29, p<0.05) and D6D (odds ratio=1.35, p<0.05), as well as low estimated D5D activity (odds ratio=0.71, p<0.001) predicted the development of metabolic syndrome (as defined by the National Cholesterol Education Program). The predictive value of D5D activity was independent of lifestyle factors (smoking, BMI and physical activity), whereas the risk associated with higher SCD-1 and D6D activities was mainly explained by obesity. Among those developing metabolic syndrome (119 out of 706) during follow-up, the proportions of fatty acids 14:0, 16:0, 16:1 (n–7), 18:1 (n–9), 18:3 (n–6) and 20:3 (n–6) were increased at baseline, while 18:2 (n–6) was decreased (p<0.05 for all).Conclusions/interpretation Serum fatty acid composition predicts the long-term development of the metabolic syndrome, and D5D activity may be particularly important in this process. Our results suggest a role of dietary fat quality in the development of metabolic syndrome, but the possibility that altered fatty acid composition, partly secondary to genetic or hormonal factors, should also be considered.     

Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.

Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase.     

The ferritin Fe2 site at the diiron catalytic center controls the reaction with O2 in the rapid mineralization pathway

Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O₂, releasing Fe(III)₂–O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln¹³⁷ and variable Asp/Ser/Ala¹⁴⁰ substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln¹³⁷ → Glu and Asp¹⁴⁰ → His, to ferritin with natural diiron substrate site variations, Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰. In Gln¹³⁷ → Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)–O product formed, ΔA₃₅₀ = 50% of wild type. In Asp¹⁴⁰ → His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰ formed diferric peroxo intermediates with variable kinetic stabilities and rates: t_1/2 varied 1- to 10-fold; k_cat varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe–active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)₂–O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues—e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides—illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.     

Comparison of fatty acid proportions in serum cholesteryl esters among people with different glucose tolerance status: the CoDAM study

Background and aimAltered fatty acid patterns in blood may be associated with insulin resistance and related disorders. We investigated whether serum proportions of cholesteryl fatty acids and desaturase activity are associated with glucose tolerance status and insulin resistance.Methods and resultsData were obtained from a cross-sectional study among 471 Dutch participants aged ≥40 years. Individual fatty acids in serum cholesteryl esters were determined and endogenous conversions by desaturases were estimated from product-to-precursor ratios. Proportions of fatty acids were compared among participants with normal glucose tolerance, impaired glucose metabolism and newly diagnosed type 2 diabetes. Partial Spearman correlation coefficients between fatty acids and homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. Adjustments were made for lifestyle and nutritional factors.The proportions of total saturated, mono-unsaturated, trans- and poly-unsaturated fatty acids did not differ significantly between groups, but several individual fatty acids did; the proportions of C18:0 and C20:3n6 were higher, whereas those of C18:1n7 and C20:4n6 were lower in participants with type 2 diabetes compared with those with normal glucose tolerance. Activity of Δ5-desaturase, that is, ratio of C20:4n6 to C20:3n6, was lower (p < 0.01) in participants with type 2 diabetes (7.4) than with normal glucose tolerance (8.4). HOMA-IR was correlated positively with Δ9-desaturase activity (r = 0.11, p < 0.01) and inversely with Δ5-desaturase activity (r = ?0.21, p < 0.01).ConclusionThe observed lower Δ5-desaturase activity in participants with type 2 diabetes and its inverse association with HOMA-IR suggest that changes in fatty-acid metabolism may play a role in the aetiology of type 2 diabetes.     

Role of FADS1 and FADS2 polymorphisms in polyunsaturated fatty acid metabolism

Tissue availability of polyunsaturated fatty acids (PUFAs) depends on dietary intake and metabolic turnover and has a major impact on human health. Strong associations between variants in the human genes fatty acid desaturase 1 (FADS1, encoding Δ-5 desaturase) and fatty acid desaturase 2 (FADS2, encoding Δ-6 desaturase) and blood levels of PUFAs and long-chain PUFAs (LC-PUFAs) have been reported. The most significant associations and the highest proportion of genetically explained variability (28%) were found for arachidonic acid (20:4n-6), the main precursor of eicosanoids. Subjects carrying the minor alleles of several single nucleotide polymorphisms had a lower prevalence of allergic rhinitis and atopic eczema. Therefore, blood levels of PUFAs and LC-PUFAs are influenced not only by diet, but to a large extent also by genetic variants common in a European population. These findings have been replicated in independent populations. Depending on genetic variants, requirements of dietary PUFA or LC-PUFA intakes to achieve comparable biological effects may differ. We recommend including analyses of FADS1 and FADS2 polymorphism in future cohort and intervention studies addressing biological effects of PUFAs and LC-PUFAs.