Lipids DOI 10.1007/s11745-011-3617-2

REVIEW

The Front-end Desaturase: Structure, Function, Evolution and Biotechnological Use

Dauenpen Meesapyodsuk • Xiao Qiu

Received: 30 July 2011 / Accepted: 26 August 2011 Ó AOCS 2011

Abstract Very long chain polyunsaturated fatty acids engineer production of these fatty acids in transgenic oil- such as arachidonic acid (ARA, 20:4n-6), eicosapentaenoic seed plants for nutraceutical markets. acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are essential Keywords Desaturases Á n-3 fatty acids Á n-6 fatty acids Á components of cell membranes, and are precursors for a Polyunsaturated fatty acids (PUFA) Á Biotechnology Á group of hormone-like bioactive compounds (eicosanoids Lipid biochemistry Á General area and docosanoids) involved in regulation of various physi- ological activities in animals and humans. The biosynthesis of these fatty acids involves an alternating process of fatty Introduction acid desaturation and elongation. The desaturation is cat- alyzed by a unique class of oxygenases called front-end A desaturase is a special type of oxygenase that can remove desaturases that introduce double bonds between the pre- two hydrogens from a hydrocarbon chain, especially from a existing double bond and the carboxyl end of polyunsatu- fatty acyl chain, catalyzing the formation of a double bond rated fatty acids. The first gene encoding a front-end in the substrate [1, 2]. Unlike normal oxygenases which desaturase was cloned in 1993 from cyanobacteria. Since directly transfer molecular oxygen to a substrate; a desat- then, front-end desaturases have been identified and char- urase uses activated molecular oxygen to abstract hydro- acterized from a wide range of eukaryotic species including gens from the substrate creating a carbon/carbon double algae, protozoa, fungi, plants and animals including bond in a fatty acid and a molecule of water [1, 3]. humans. Unlike front-end desaturases from bacteria, those According to their regioselectivity, desaturases are typ- from eukaryotes are structurally characterized by the ically categorized as Dx desaturase that introduces a double presence of an N-terminal cytochrome b5-like domain bond at position x referred to from the carboxyl end of a fused to the main desaturation domain. Understanding the fatty acid; or xy desaturase that introduces a double bond structure, function and evolution of front-end desaturases, at position y referred to from the methyl end [4–6]. In as well as their roles in the biosynthesis of very long chain addition, desaturases can additionally be labelled as m?zor polyunsaturated fatty acids offers the opportunity to m-z desaturases. The m?z desaturase introduces a double bond at z carbons after the pre-existing double bond m,[7] i.e. towards the methyl end, while the m-z desaturase can D. Meesapyodsuk Á X. Qiu introduce a double bond at z carbons before the pre- Department of Food and Bioproduct Sciences, existing double bond m, i.e. towards the carboxyl end. An University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N5A8, Canada example of a Dx desaturase is the acyl-ACP D9 desaturase from plants, a soluble enzyme introducing a first D9 double D. Meesapyodsuk Á X. Qiu (&) bond into saturated palmitoyl-ACP or stearoyl-ACP [8, 9]. Plant Biotechnology Institute, National Research The membrane-bound x3 desaturase from nematode Cae- Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N0W9, Canada norhabditis elegans is an example of a xy desaturase that e-mail: [email protected] inserts an x3 double bond into a polyunsaturated fatty acid 123 Lipids

[10, 11]. An example of m?3 desaturase is the Claviceps purpurea ‘‘D12’’ desaturase that has a preference for introducing double bonds at the D12 position, three carbons after the pre-existing double bond at the ninth position [7], while the D4 desaturase from Thraustochytrium is cata- lytically a m-3 desaturase and can only introduce a double bond at position 4 which is three carbons before the pre- existing double bond at the seventh position [12]. Based on the position of the double bond insertion rel- ative to a pre-existing double bond in a fatty acyl chain, desaturases can also be referred to as front-end desaturases or methyl-end desaturases[13]. Unsaturated fatty acids are essential for all living species in which the initial de novo fatty acid synthesis generally results in production of sat- urated fatty acids with 18 carbons or 16 carbons in length. The first double bond is often inserted at approximately the middle position of a fatty acid chain. Fatty acids with different chain length and double bond position are gen- erated later by various fatty acid modifying enzymes, such as elongases and desaturases. A methyl-end desaturase introduces a double bond between the pre-existing double Fig. 1 Front-end desaturases involved in the biosynthesis of very bond and the methyl-end, while a front-end desaturase long chain polyunsaturated fatty acids. The dotted arrow indicates Sprecher’s pathway for DHA biosynthesis inserts a double bond between the pre-existing double bond and the carboxyl end of a fatty acid [14]. Commonly-found to dihomo-gamma-linolenic acid (DGLA, 20:3-8,11,14) membrane-bound xy and m?z desaturases such as ‘‘D12’’, and eicosatetraenoic acid (ETA, 20:4-8,11,14,17), which ‘‘D15’’ and x3 desaturases in plants are examples of are then desaturated by a second front-end desaturase—the methyl-end desaturases, while widely spread m-z desatu- D5 desaturase giving rise to arachidonic acid (ARA, 20:4- rases in microorganisms such as D4, D5, D6 and D8 5,8,11,14) and eicosapentaenoic acid (EPA, 20:5-5,8,11, desaturases belong to front-end desaturases. 14,17), respectively. EPA is elongated to docosapentaenoic Although both methyl-end desaturase and front-end acid (DPA, 22:5-7,10,13,16,19) which is then desaturated desaturase are involved in the biosynthesis of very long by a third front-end desaturase—the D4 desaturase, giving chain polyunsaturated fatty acids, their occurrence in living docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) in the species is not identical. The former is widely present in x3 pathway. However, mammals including humans lack plants and microorganisms, while the latter mostly occur in the D4 desaturase. Biosynthesis of DHA in mammals takes animals and microorganisms, although certain types of ‘‘the retro-conversion pathway’’ [19] which involves two front-end desaturases have been identified in a small num- rounds of chain elongation of EPA and another D6 desat- ber of higher plants, such as borage [15], echium [16] and uration on the elongated product, followed by a single conifers [17]. Higher animals including humans lack the 2-carbon chain shortening of the D6 desaturated product in methyl-end desaturase such as ‘‘D12’’, ‘‘D15’’ and x3 the peroxisome, giving DHA. The D8 desaturase is another desaturase [18]. Consequently, they cannot synthesize lin- front-end desaturase involved in the biosynthesis of very oleic acid (LA, 18:2-9,12) and linolenic acid (ALA, 18:3- long chain polyunsaturated fatty acids [20–22]. This 9,12,15) from oleic acid (OA, 18:1-9), the two essential desaturase works on a branching pathway of the biosyn- fatty acids that have to be acquired from the diet. LA and thesis, introducing a D8 double bond into elongated prod- ALA are precursors for the biosynthesis of very long chain ucts LA or ALA, i.e. 20:2-11,14 or 20:3-11-14,17, giving polyunsaturated fatty acids such as arachidonic acid (20:4n- rise to DGLA and ETA, respectively, which can then be 6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and doco- desaturated by a D5 desaturase, giving ARA and EPA as sahexaenoic acid (22:6n-3, DHA). As shown in Fig. 1,to described above (Fig. 1). The individual front-end desatu- synthesize these fatty acids, LA and ALA are desaturated by rases are described below. a first front-end desaturase—the D6 desaturase, introducing a D6 double bond into the substrates giving gamma-lino- D6 Desaturase lenic acid (GLA, 18:3-6,9,12) in the x6 pathway, and stearidonic acid (SDA, 18:4-6,9,12,15) in the x3 pathway, The first D-6 desaturase gene was cloned in 1993 from the respectively. GLA and SDA are elongated by a D6 elongase cyanobacterium Synechocystis using a gain-of-function 123 Lipids expression approach [23]. The protein sequence of this first who expressed the Synechocystis D6 desaturase under the front-end desaturase shows a similarity to other acyl-lipid control of a constitutive CaMV 35S promoter in tobacco. desaturases with different regioselectivity from cyanobac- Transgenic tobacco carrying the gene produced very small teria [23]. Like its homologous sequences, the D6 desat- amounts of GLA and SDA in leaves, but not in seeds [15, urase from Synechocystis does not contain a cytochrome 41]. That the prokaryotic D6 desaturase did not work well b5-like domain as was later found in eukaryotic D6 desat- in plants might be due to incompatible cofactors required urases. A similar D6 desaturase was later cloned from for the desaturation (see below). Production of high levels another cyanobacterium Spirulina platensis [24]. In 1997, of GLA and SDA, two D6 desaturated fatty acids, in the first eukaryotic D6 desaturase gene was cloned from transgenic oilseeds has since been achieved by several borage plant by two independent groups [15, 25]. Unlike other groups using eukaryotic D6 desaturases [42]. The bacterial D6 desaturases, the D6 desaturase encoded by this seed-specific expression of a fungal D6 desaturase and D12 gene is a modular protein that has a cytochrome b5-like desaturase from Mortierella alpina produced up to 43% domain fused to the N-terminus of the main desaturation GLA in seeds of Brassica napus [43]. A similar level of domain. This modular structure was later observed to be a GLA was produced in B. juncea seeds by expressing a feature of all the D6 desaturases isolated from eukaryotes single D6 desaturase from fungus Pythium irregulare [32]. including algae [26–28], moss [29], plants [15, 16], fungi Seed expression of a B. napus D15 desaturase gene with [30–32], animals [33, 34] and humans [35]. The existence M. alpina D6 and D12 desaturases resulted in production of of an N-terminal cytochrome b5-like domain is not exclu- SDA accounting for 16–23% of the total fatty acids [44]. sive to this front-end desaturase, a similar motif has been Co-expression of a borage D6 desaturase and an Arabid- also observed in other front-end desaturases such as D5, D8 opsis D15 desaturase in soybean produced up to 30% of and D4 desaturases and some sphingolipid desaturases SDA [45]. Very recently, a very high level of GLA (more from eukaryotes [36]. Beside the cytochrome b5-like than 60%) in transgenic safflower oilseeds was achieved domain at the N-terminus, the rest of the D6 desaturase using a D6 desaturase from Saprolegnia diclina [46]. sequences have sequence similarity to methyl-end desatu- rases with characteristics of three highly conserved D5 Desaturase histidine-rich motifs, i.e. H–X3-4–H, H–X2-3-H–H and H/Q–X2-3-H–H. However, the first histidine of the third The first D5 desaturase gene was cloned in 1998 from the motif in the front-end desaturase is usually replaced by fungus M. alpina by two independent groups almost glutamine [15]. Conversion of this residue back to histidine simultaneously using sequence information of the front-end resulted in loss of the activity, implying this residue might D6 desaturases from borage for degenerate RT-PCR [47, be very important for the structural configuration of this 48]. Since then, many D5 desaturases have been cloned modular type of desaturases for front-end desaturation [37]. from diverse species including lower plants [49, 50], ani- The function of D6 desaturases isolated from prokary- mals [51] including human [52], fungi [48, 53], moss [54], otes and eukaryotes was mostly established by their protozoa [55] and algae [28, 56]. Like D6 desaturases from expression either in yeast (Saccharomyces cerevisiae or eukaryotes, the D5 desaturase comprises a cytochrome

Pichia pastoris) or in plants. Functional enzymatic studies b5-like domain fused at the N-terminus of the sequence. have showed that most of D6 desaturases isolated from The function of the D5 desaturases was mostly estab- eukaryotes can use both LA and ALA as substrates effec- lished by expressing the genes in yeast. Like the D6 tively at the similar level. However, some D6 desaturases desaturase, D5 desaturase can use two substrates, both x3 showing a preference for ALA have also been reported (ETA) and x6 (DGLA) are desaturated almost equally [38]. A bifunctional D6 desaturase was identified in effectively, producing EPA and ARA, respectively. How- Ceratodon purpureus that can convert the D6 double bond ever, besides the two major substrates, some D5 desaturases it creates to a triple bond [39]. A human D6 desaturase was are active on LA and ALA, producing polymethylene- found to have the capability of introducing a D6 double interrupted D5 desaturated fatty acids such as pinolenic acid bond into two distinct substrates with different chain (18:3-5,9,12), which was found naturally in seeds of some lengths, i.e. 18:2-9,12 and 24:5-9,12,15,18,21 [40]. The conifer trees [53]. A D5 desaturase from zebrafish also observation of the D6 desaturation of the latter substrate carries D6 desaturase activity at the similar level, i.e. it is provides the important evidence supporting the retro-con- bifunctional [57]. version pathway that requires the desaturation step for Various D5 desaturase have been used to transform DHA biosynthesis in humans. plants for transgenic production of D5 desaturated poly- The first published transgenic production of GLA, a D6 unsaturated fatty acids. Abbadi et al. [58] (2004) intro- desaturated fatty acid in the x6 pathway, in plants using a duced a D5 desaturase from M. alpina along with a D6 D6 desaturase was reported by Reddy and Thomas (1996) desaturase and D6 elongase from Physcomitrella patens 123 Lipids into tobacco and flax, which led to the accumulation of have met with only limited success; a level of DHA in small amounts of ARA and EPA in transgenic seeds. An transgenic oilseeds that is commercially viable has not acyl-CoA D5 desaturase from an alga, along with other been achieved. genes, was also used to transform Arabidopsis resulting in production a small amount of D5 desaturated fatty acids in D8 Desaturase seeds [27]. Recently a high level of EPA in transgenic Brassica carinata was reported using a Thraustochytrium The first D-8 desaturase gene was cloned in 1999 using D5 desaturase along with a D6 desaturase from Pythium degenerate RT-PCR amplification from the protist Euglena irregulare and an elongase from Thraustochytrium [59]. It gracilis [20]. The deduced protein sequence is highly appears that D5 desaturase from fungal species works very similar to the D6 desaturase from Caenorhabditis elegans effectively when expressed in plants. with a cytochrome b5-like domain at the N-terminus. Functional analysis of this gene in yeast shows the desat- D4 Desaturase urase introduces a D8 double bond in 20:2-11,14 and 20:3- 11,14,17 which could be produced by the elongation of LA The D4 desaturase gene was first cloned in 2001 using the and ALA [69], giving 20:3-8,11,14 and 20:4-8,11,14,17, degenerate RT-PCR approach from Thraustochytrium,a respectively. Since the discovery of the D8 desaturases, this single cellular Thraustochytrid that can accumulate a high branching pathway has drawn much attention because it level of DHA [12]. In 1991, 10 years previously, Sprecher provides an alternative to the traditional D6 desaturation and co-workers published their systematic work on the pathway for the biosynthesis of very long chain polyun- biosynthesis of DHA which suggested that DHA biosyn- saturated fatty acids. Homologous D8 desaturases have thesis in mammals occurs independently of a D4 desaturase now been isolated from protozoan [20–22], mammals [70] and involves retro-conversion of a 24-carbon D6 fatty acid and marine algae [71]. through a controlled b-oxidation [60]. According to this Use of the D8 desaturase was first attempted, along with pathway, the intermediate C24 polyunsaturated fatty acid a D9 elongase from Isochrysis galbana, and a D5 desat- required for DHA synthesis would be expected to be urase from M. alpina for production of D5 desaturated fatty observed in Thraustochytrium. However, when the fatty acids in vegetative tissues of Arabidopsis [72]. The acid profile of Thraustochytrium was examined, no poly- branching pathway via this front-end desaturase was per- unsaturated fatty acids more than 22 carbons in length were ceived to be advantageous over the traditional D6 desat- found. Therefore, DHA biosynthesis in Thraustochytrium urase pathway in overcoming the elongation bottleneck. was suggested not to follow the Sprecher’s pathway, but However, the D8 desaturase from Euglena appeared to instead to involve a D4 desaturase that might have a primary have low activity in plants. As such, only a very low structure similar to other eukaryotic front-end desaturases amount of ARA and EPA was produced in oilseed crops such as the D6 desaturase in borage and the D5 desaturase in when this desaturase along with other elongases and de- M. alpina. The D4 desaturase gene was thus isolated from saturases was co-expressed (Qiu et al. unpublished data). Thraustochytrium using the degenerate RT-PCR strategy Recently, a new D8 desaturase from Pavlova salina,aD9 and it indeed encodes a fusion protein with D4 desaturation elongase from I. galbana and a D5 desaturase from activity introducing a D-4 double bond in docosapentaenoic P. salina were co-expressed in transgenic Arabidopsis acid (22:5n-3) and docosatetraenoic acid (22:4n-6), thaliana and Brassica napus, respectively, resulting in respectively. Since then, a few other D4 desaturases have production of a relatively high level of ARA (20%) in been isolated from protists [61, 62] and microalgae [63, 64] Arabidopsis seeds and a low level of EPA (approximately using the sequence information of this desaturase. It is 3%) in B. napus seeds [73]. noteworthy that the first D4 desaturase of vertebrate animals involved in the DHA biosynthesis was recently identified in teleost fish (Signus canaliculatus)[65]. Acyl Carrier Substrate Specificity of Front-end The discovery of the first D4 desaturase in microor- Desaturases ganisms provides a simple pathway for DHA biosynthesis and suggests the possibility of producing this important Desaturases can be classified as acyl-CoA desaturases, fatty acid in heterologous systems, especially in plants, in a acyl-ACP desaturases and acyl-lipid desaturases based on cost-effective way. Indeed, several laboratories worldwide the acyl-carrier they use as substrate. Acyl-ACP desatu- have since actively pursued reconstitution of this simple rases have only been found in the lumen of plastids in pathway to produce DHA in plants, such as in soybean plants where de novo biosynthesis of fatty acids occurs. [66], Brassica juncea [67] and Arabidopsis thaliana [68]. Saturated fatty acids freshly synthesized in the plastids However, so far, these efforts to produce DHA in oilseeds which are still linked to acyl carrier protein are the 123 Lipids substrate for this type of desaturase [74]. Acyl-CoA de- ferredoxin reductase and NADPH as the electron transport saturases are usually membrane-bound and located in the system [2]. The front-end desaturases located in endo- endoplasmic reticulum of eukaryotes using fatty acid plasmic reticulum of eukaryotes such as acyl-CoA desat- linked to Coenzyme A as substrate, while acyl-lipid de- urases from animals and phospholipid desaturases from saturases introduce double bonds into fatty acids linked to plants and fungi are the eukaryotic type that utilizes complex lipid molecules such as glycoglycerolipid, phos- cytochrome b5, cytochrome b5 reductase and NADH as the phoglycerolipid and sphingolipids [2]. Depending on the electron transport system [79]. As mentioned above, most origin, front-end desaturases can be either acyl-CoA of front-end desaturases, except for the ones from cyano- desaturases or acyl-lipid desaturases. All known mamma- bacteria [23, 24] are fusion proteins with a cytochrome b5- lian desaturases are believed to be acyl-CoA desaturases like domain at the N-terminus. This structure suggests that

[14], thus front-end desaturases from higher animals would all these front-end desaturase would use cytochrome b5 as be predicted to use acyl-CoA as substrates. In plants, evi- electron donor for the desaturation. It is noteworthy that the dence on the substrate specificity of front-end desaturase existence of a cytochrome b5 fusion is also observed in a was provided by Stymne and colleagues using borage D6 yeast D9 desaturase where the cytochrome b5-like domain desaturation as an example where [14C]-linoleoyl phos- is fused at the C-terminus [80]. Deletion of the C-terminal phatidylcholine was desaturated to radioactive c-linolenoyl cytochrome b5 domain of the yeast D9 desaturase resulted phosphatidylcholine in developing seeds, indicating that in complete loss of enzymatic activity [81]. Similarly, the substrate for the linoleate D6-desaturase was phos- when expressed in the endogenous cytochrome b5-dis- phatidylcholine. In addition, they also showed that the D6 rupted strain or the wild type yeast strain, the borage D-6 desaturase was positionally specific to linoleate at the sn-2 desaturase is able to functionally introduce a D6 double position of phosphatidylcholine [75, 76]. Recently, the acyl bond into the substrate, whereas, deletion of the N-terminal carrier substrate specificity of front-end desaturases was cytochrome b5-like domain of the desaturase resulted in the systematically examined in a yeast system using a variety loss of the function [82], indicating that cytochrome b5-like of D6 and D5 desaturases by Heinz and colleagues. domain is an essential part of the enzyme such that the free

Detailed analysis of desaturated products in acyl-CoA, cytochrome b5 could not substitute the electron transport phospholipid and neutral lipid pools during a time course function. Spirulina platensis D6 desaturase is a smaller showed that front-end desaturases from lower plants, fungi, polypeptide which does not contain the cytochrome b5-like worms and algae belong to acyl-lipid desaturases where the domain. When expressed in Escherichia coli with supple- desaturation occurs predominately at the sn-2 position of mentation of ferredoxin, the transformant produced GLA phosphatidylcholine. On the other hand, a human D6 from exogenous LA. However, when expressed in yeast, desaturase tested uses linoleoyl-CoA as the substrate [26, Spirulina D6 desaturase N-fused or co-expressed with the

77]. Since then, a few acyl-CoA front-end desaturases have cytochrome b5 from Mucor rouxii produced GLA from LA been identified from microalgae using the same approach [83]. This result indicates that the prokaryotic front-end [27, 78]. However, it is worthwhile to point out that all desaturases may not have a strict requirement for a particular these assignments for acyl carrier specificity are solely electron transport system. The ferredoxin requirement of based on the in vivo experiment where the yeast strain the prokaryotic D6 desaturase can be complemented by the expressing a heterologous front-end desaturase was sup- corresponding eukaryotic electron donor, cytochrome b5. plied with substrates and presence of desaturated products However, for eukaryotic front-end desaturases, the require- in different lipid classes in a time course was used to assign ment for electron donor appears to be more stringent that the substrate form. Although this in vivo approach can deletion of cytochrome b5 domain or mutation of the provide useful information on the likely acyl carriers used heme-binding motif in the cytochrome b5 domain of front- by front-end desaturases, direct evidence such as that end desaturases totally eliminates the activity [82, 84]. obtained from in vitro assays is needed for conclusive determination of the substrate form of the desaturases. Structural Determinants for Regioselectivity of Front-end Desaturases Electron Donors of Front-end Desaturases The regioselectivity of a desaturase refers to the specific The front-end desaturation reaction, like other oxygenation positioning of a double bond in a fatty acyl chain. Gener- reactions, is an aerobic process requiring molecular oxygen ally, front-end desaturases, regardless of their origin, have and an electron donor for the oxidation. Like acyl-ACP high regioselectivity and substrate specificity which are desaturase and glycolipid desaturases from plastids of determined by the structure of the desaturase proteins. plants, front-end desaturases in cyanobacteria are the pro- However, due to lack of information of three-dimensional karyotic type of acyl-lipid desaturases that use ferredoxin, structures of front-end desaturases, there is little 123 Lipids information available on the exact relationship between the according to deuterium kinetic isotope effects associated structure and function of these enzymes. Nevertheless, a with the C–H bond cleavages [93]. Actual stereospecific model previously proposed [11] for the methyl-end desat- syn-dehydrogenation might be controlled by the geometry urases can shed some light on the structure–function rela- of the hydrophobic pocket that is in the proximity of the tionship of the front-end desaturase. In this model, di-iron center of the desaturase [79, 94]. However, the structural determinants for substrate specificity and regi- detailed mechanism underlying the front-end desaturation oselectivity include a head group binding domain or a remains to be elucidated. methyl group-binding domain, a pre-existing double bond binding domain, and the active site. From this model, we Evolution of Front-end Desaturases would presume that the substrate specificity and regiose- lectivity of front-end desaturases might be controlled by The evolution of desaturases as a whole has been thor- more than one domain. Targeted mutagenesis of amino oughly reviewed by Heinz and colleagues [79]. According acid residues that are present in close proximity to the to the solubility in an aqueous environment, desaturases histidine boxes of Mucor rouxii D6 desaturase revealed that can be classified into two major groups, soluble desaturases some of these residues are involved in substrate binding and membrane-bound desaturases. Each group of desatu- [85]. Using domain swapping, Napier and colleagues rases possesses characteristic features in their primary showed that the regions of first two membrane-spanning structure and is believed to have evolved independently helices and carboxyl terminus of borage front-end desatu- [79]. Front-end desaturases are membrane-bound enzymes rases are important for the substrate binding and regiose- with a wide range of substrate selectivity and high regi- lectivity [86]. Despite these studies, our understanding of oselectivity and/or stereospecificity. This type of desatu- the individual functional domains of front-end desaturases rases is recalcitrant to biochemical purification, thus no still remains very limited. information is available on the three dimensional structure of the desaturases. In the past decade, the number of front- The Catalytic Mechanism of Front-end Desaturases end desaturase sequences that have been functionally characterized has grown rapidly with the advance of Like other desaturases, the front-end desaturases belong to molecular cloning and genome sequencing technologies. a group of metalloenzymes that activate the molecular This has provided opportunities for an evolutionary anal- oxygen using non-heme di-iron active sites to abstract two ysis of the front-end desaturases to elucidate their phylo- hydrogens from fatty acids resulting in the introduction of a genetic origin and catalytic diversity based on amino acid front-end double bond [87]. Non-heme iron-containing sequence information. As shown in Fig. 2, front-end oxygenases have been widely found in nature. They cata- desaturases in microorganisms, plants and animals are lyze not only fatty acid desaturation, but also a wide range evolutionarily distinct from methyl-end desaturases from of other oxygenation reactions [87, 88]. For instance, non- cyanobacteria and plants. In another words, the evolution heme iron-containing alkane monooxygenase catalyzes of front-end desaturases has undertaken a path different hydroxylation of alkane producing alcohol, which has been from that of the methyl-end desaturases. Front-end desat- the focus of recent research on petroleum contamination urases selected from a wide range of organisms including remediation [89]. Non-heme iron-containing lipoxygenase cyanobacteria, algae, protozoa, moss, fish, plants and that catalyzes the insertion of a molecular oxygen into the mammals can be phylogenetically grouped into three main (1Z,4Z)-pentadiene of a polyunsaturated fatty acid pro- clades. The first clade consists of animal front-end desat- ducing hydroxyperoxide, a key precursor for a variety of urases including those from fish and humans. The second oxylipins involved in the response to biotic and abiotic clade comprises front-end desaturases mainly from plants, stresses [90]. Catechol dioxygenase is another non-heme worms and fungi. The third clade contains front-end iron-containing enzyme that converts 3,4-dihydroxybenzo- desaturase mainly from algae, fungi and cyanobacteria. ate to b-carboxy-cis,cis-muconate [91]. Although these The cyanobacterial front-end desaturases grouped with oxygenases have distinct primary structure in protein those of algae and fungi, but not with the methyl end sequences, and catalyze different enzymatic reactions, the (‘‘D12’’) desaturases from their own species, reaffirms that catalytic mechanism is found to be similar by using the front-end desaturases might take an evolutionary path dif- oxo-ferric intermediate in the catalytic reaction [1, 88]. The ferent from the methyl-end desaturases. The prototype of di-iron catalytic center is believed to be coordinated by prokaryotic D6 desaturase might be the progenitor of all the three conserved histidine-rich motifs [92]. Abstraction of front-end desaturases in eukaryotes, even though their two hydrogens by front-end desaturases usually takes place primary structures are quite different. In addition, it could in a stepwise manner as syn-elimination with the initial also be seen from the phylogenetic tree that front-end oxidation site close to the carbon at the carboxyl end desaturases are not always clustered according to their 123 Lipids

Fig. 2 Phylogenetic analysis of front-end desaturases. Amino acid alignment was used to generate a rooted phylogenetic tree using the sequences retrieved from NCBI by their accession number were neighbor joining method and visualized by Treeview aligned using ClustalW using default parameters. The resulting regioselectivity and origin. For instance, D5 and D6 evolutionary point of view, front-end desaturases with D5, desaturases from C. elegans which are tightly grouped D8 and D4 regioselectivity in eukaryotes might have together belong to the clade of front-end desaturases evolved independently from the progenitor D6 desaturase including D8 desaturases from protist, D6 desaturases from through multiple independent gene duplication events and fungi and plants. On the other hand, the Mortierella D6 diversification. It is noteworthy that we should not rule out desaturase forms a clade with other D6 desaturases from the other possibility that eukaryotic front-end desaturases algae and plants, while the Mortierella D5 desaturase might have evolved from their sphingolipid desaturases as belongs to the clade of front-end desaturases with D4, D5, the latter occur more widely and are very closely related to D6 and D8 regioselectivity from algae and fungi. From an the front-end desaturases. Since sphingolipid desaturases 123 Lipids do not belong to front-end desaturase, they are not wild fish and oleaginous fungi. Oils from marine fish for described here. these fatty acids are already over-exploited, which results in dramatic reductions of fish populations in ocean. In addition, possible contamination in fish oil with heavy Concluding Remarks metals and toxins is increasingly becoming a concern. Oil from oleaginous fungi is expensive due to the high cost of Front end desaturases are remarkable for their structural fungal culture and oil extraction. Therefore, the scientific similarity and functional diversity. Almost all the front-end community and commercial entities are currently under desaturases except for cyanobacterial ones are modular intensive pressure to explore alternatives to these fatty proteins containing a cytochrome b5-like domain at the acids. Transgenic production of very long chain polyun- N-terminus and a desaturation domain at the C-terminus. saturated fatty acids in plants using genes encoding the Three conserved histidine-rich motifs which are believed to front-end desaturases and elongases cloned from microal- be responsible for di-iron binding at the catalytic center are gae and fungi have been viewed as an attractive alternative located at the C-terminal domain. All of front-end desat- source for these fatty acids. The biosynthetic pathways of urases from eukaryotes share a similar hydrophobicity these fatty acids have been successfully reconstituted in profile, which indicates a common membrane topology plants independently by several groups worldwide [97, 98]. [86]. Phylogenetic analysis of front-end desaturases pro- A high level of EPA and ARA has already been achieved in vides very interesting insights into the structure–evolution transgenic oilseed crops, which provide great opportunity relationships of these desaturases. Substrate specificity and for nutraceutical markets for these fatty acids as dietary regioselectivity and catalytic mechanisms of eukaryotic supplements. However, an economically viable level of front-end desaturases involve not only the structure feature DHA in transgenic plants has not been realized due to the of the desaturase, but also substrate features such as fatty metabolic bottleneck of acyl trafficking in the complex acid chain length, existing double bonds and acyl carrier pathways involving too many genes [58, 67] comprising [11, 95]. three to four of those for front-end desaturases. Our fur- Very long chain polyunsaturated fatty acids are syn- ther understanding of the mechanism underlying the thesized by a series of front-end desaturases and elongases structure, function and evolution of front-end desaturases which play important roles in the integrity and function of is vital for us to reach the goal—production of a high level biological membranes in eukaryotes. Perceivably, their of very long chain x3 fatty acids in transgenic oilseed biosynthesis in the cells should be subjected to the tight crops for nutraceutical and functional food markets control through expression regulation of these enzymes. [99–102]. However, so far, there has been no information available on how the expression of these desaturases and elongases is Acknowledgments We thank Drs. Mark Smith and Patrick Covello regulated. There is evidence that the biosynthesis of for their critical reading of this manuscript. We also wish to thank the authors for their wonderful works referred here. As there are a large unsaturated fatty acids in bacteria and human is achieved number of publications in this field, we apologize to those authors by the feedback control of transcriptional regulation of whose works are relevant but might have been overlooked and thus fatty acid desaturase genes, mediated through signalling not included in this review. pathways activated by sensors embedded in cellular membranes, in response to environmental factors [96]. The References regulation of front-end desaturases is likely to adopt the similar strategy. If so, how is the transcriptional regulatory 1. Shanklin J, Cahoon EB (1998) Desaturation and related modi- control achieved for front-end desaturase genes? Answer- fications of fatty acids. Annu Rev Plant Physiol Plant Mol Biol ing this question will be an attractive target for the future 49:611–641 2. Los DA, Murata N (1998) Structure and expression of fatty acid research of front-end desaturases. desaturases. Biochim Biophys Acta 1394:3–15 Very long chain x3 polyunsaturated fatty acids are 3. Buist PH (2004) Fatty acid desaturases: selecting the dehydro- essential for human health and well-being. Numerous genation channel. Nat Prod Rep 21:249–262 randomized, placebo-controlled and double blind trials 4. Yadav NS, Wierzbicki A, Aegerter M, Caster CS, Perez-Grau L, Kinney AJ, Hitz WD, Booth JR Jr, Schweiger B, Stecca KL have shown that dietary supplementation of these fatty (1993) Cloning of higher plant omega-3 fatty acid desaturases. acids can provide protection against many common chronic Plant Physiol 103:467–476 diseases such as cardiovascular diseases, diabetics and 5. Hitz WD, Carlson TJ, Booth JR Jr, Kinney AJ, Stecca KL, inflammatory disorders. Moreover, they also play impor- Yadav NS (1994) Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobac- tant roles in maintaining the function or enhancing per- terium. Plant Physiol 105:635–641 formance of the eyes and brain. Unfortunately, the current 6. Reed DW, Schafer UA, Covello PS (2000) Characterization of dietary source for these fatty acids is limited to oils from the Brassica napus extraplastidial linoleate desaturase by 123 Lipids

expression in Saccharomyces cerevisiae. Plant Physiol Synechocystis sp. strain PCC 6803 by gain-of-function expres- 122:715–720 sion in Anabaena sp. strain PCC 7120. Plant Mol Biol 7. Meesapyodsuk D, Reed DW, Covello PS, Qiu X (2007) Primary 22:293–300 structure, regioselectivity, and evolution of the membrane- 24. Deshnium P, Paithoonrangsarid K, Suphatrakul A, Meesapyod- bound fatty acid desaturases of Claviceps purpurea. J Biol suk D, Tanticharoen M, Cheevadhanarak S (2000) Temperature- Chem 282:20191–20199 independent and -dependent expression of desaturase genes in 8. Fox BG, Shanklin J, Somerville C, Munck E (1993) Stearoyl- filamentous cyanobacterium Spirulina platensis strain C1 acyl carrier protein delta 9 desaturase from Ricinus communis is (Arthrospira sp. PCC 9438). FEMS Microbiol Lett 184:207–213 a diiron-oxo protein. Proc Natl Acad Sci USA 90:2486–2490 25. Thomas TL, Reddy AS, Nuccio M, Nunberg AN, Freyssinet GL 9. Fox BG, Shanklin J, Ai J, Loehr TM, Sanders-Loehr J (1994) (1997) Production of gamma-linolenic acid by a delta6-desat- Resonance Raman evidence for an Fe-O-Fe center in stearoyl- urase. U.S. Patent 4,736,866 ACP desaturase. Primary sequence identity with other diiron- 26. Domergue F, Abbadi A, Zahringer U, Moreau H, Heinz E oxo proteins. Biochemistry 33:12776–12786 (2005) In vivo characterization of the first acyl-CoA Delta6- 10. Spychalla JP, Kinney AJ, Browse J (1997) Identification of an desaturase from a member of the plant kingdom, the microalga animal omega-3 fatty acid desaturase by heterologous expres- Ostreococcus tauri. Biochem J 389:483–490 sion in Arabidopsis. Proc Natl Acad Sci USA 94:1142–1147 27. Hoffmann M, Wagner M, Abbadi A, Fulda M, Feussner I (2008) 11. Meesapyodsuk D, Reed DW, Savile CK, Buist PH, Ambrose SJ, Metabolic engineering of omega3-very long chain polyunsatu- Covello PS (2000) Characterization of the regiochemistry and rated fatty acid production by an exclusively acyl-CoA-depen- cryptoregiochemistry of a Caenorhabditis elegans fatty acid dent pathway. J Biol Chem 283:22352–22362 desaturase (FAT-1) expressed in Saccharomyces cerevisiae. 28. Domergue F, Lerchl J, Zahringer U, Heinz E (2002) Cloning and Biochemistry 39:11948–11954 functional characterization of Phaeodactylum tricornutum front- 12. Qiu X, Hong H, MacKenzie SL (2001) Identification of a Delta end desaturases involved in eicosapentaenoic acid biosynthesis. 4 fatty acid desaturase from Thraustochytrium sp. involved in Eur J Biochem 269:4105–4113 the biosynthesis of docosahexanoic acid by heterologous 29. Girke T, Schmidt H, Zahringer U, Reski R, Heinz E (1998) expression in Saccharomyces cerevisiae and Brassica juncea. Identification of a novel delta 6-acyl-group desaturase by targeted J Biol Chem 276:31561–31566 gene disruption in Physcomitrella patens. Plant J 15:39–48 13. Aitzetmuller K, Tsevegsuren N (1994) Seed fatty acid ‘‘front- 30. Laoteng K, Mannontarat R, Tanticharoen M, Cheevadhanarak S end’’ desaturases and chemotaxonomy-a case study in the (2000) Delta6-desaturase of Mucor rouxii with high similarity to Ranunculaceae. J Plant Physiol 143:538–543 plant delta 6-desaturase and its heterologous expression in 14. Nakamura MT, Nara TY (2004) Structure, function, and dietary Saccharomyces cerevisiae. Biochem Biophys Res Commun regulation of delta6, delta5, and delta9 desaturases. Annu Rev 279:17–22 Nutr 24:345–376 31. Sakuradani E, Kobayashi M, Shimizu S (1999) Delta6-fatty acid 15. Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, desaturase from an arachidonic acid-producing Mortierella Christie WW, Shewry PR, Napier JA (1997) Expression of a fungus. Gene cloning and its heterologous expression in a fun- borage desaturase cDNA containing an N-terminal cytochrome gus, Aspergillus. Gene 238:445–453 b5 domain results in the accumulation of high levels of delta6- 32. Hong H, Datla N, Reed DW, Covello PS, MacKenzie SL, Qiu X desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci (2002) High-level production of gamma-linolenic acid in USA 94:4211–4216 Brassica juncea using a delta6 desaturase from Pythium irreg- 16. Garcia-Maroto F, Garrido-Cardenas JA, Rodriguez-Ruiz J, ulare. Plant Physiol 129:354–362 Vilches-Ferron M, Adam AC, Polaina J, Alonso DL (2002) 33. Zheng X, Tocher DR, Dickson CA, Bell JG, Teale AJ (2005) Cloning and molecular characterization of the delta6-desaturase Highly unsaturated fatty acid synthesis in vertebrates: new from two echium plant species: production of GLA by heter- insights with the cloning and characterization of a delta6 ologous expression in yeast and tobacco. Lipids 37:417–426 desaturase of Atlantic salmon. Lipids 40:13–24 17. Piispanen R, Saranpaa P (2002) Neutral lipids and phospholipids 34. Seiliez I, Panserat S, Kaushik S, Bergot P (2001) Cloning, tissue in Scots pine (Pinus sylvestris) sapwood and heartwood. Tree distribution and nutritional regulation of a Delta6-desaturase- Physiol 22:661–666 like enzyme in rainbow trout. Comp Biochem Physiol B Bio- 18. Pereira SL, Leonard AE, Mukerji P (2003) Recent advances in the chem Mol Biol 130:83–93 study of fatty acid desaturases from animals and lower eukaryotes. 35. Cho HP, Nakamura MT, Clarke SD (1999) Cloning, expression, Prostaglandins Leukot Essent Fatty Acids 68:97–106 and nutritional regulation of the mammalian Delta-6 desaturase. 19. Sprecher H, Luthria DL, Mohammed BS, Baykousheva SP J Biol Chem 274:471–477 (1995) Reevaluation of the pathways for the biosynthesis of 36. Sperling P, Zahringer U, Heinz E (1998) A sphingolipid desat- polyunsaturated fatty acids. J Lipid Res 36:2471–2477 urase from higher plants. Identification of a new cytochrome b5 20. Wallis JG, Browse J (1999) The Delta8-desaturase of Euglena fusion protein. J Biol Chem 273:28590–28596 gracilis: an alternate pathway for synthesis of 20-carbon poly- 37. Sayanova O, Beaudoin F, Libisch B, Castel A, Shewry PR, unsaturated fatty acids. Arch Biochem Biophys 365:307–316 Napier JA (2001) Mutagenesis and heterologous expression in 21. Sayanova O, Haslam R, Qi B, Lazarus CM, Napier JA (2006) yeast of a plant Delta6-fatty acid desaturase. J Exp Bot The alternative pathway C20 Delta8-desaturase from the non- 52:1581–1585 photosynthetic organism Acanthamoeba castellanii is an atypi- 38. Sayanova OV, Beaudoin F, Michaelson LV, Shewry PR, Napier cal cytochrome b5-fusion desaturase. FEBS Lett 580:1946–1952 JA (2003) Identification of primula fatty acid delta 6-desaturases 22. Venegas-Caleron M, Beaudoin F, Sayanova O, Napier JA with n-3 substrate preferences. FEBS Lett 542:100–104 (2007) Co-transcribed genes for long chain polyunsaturated fatty 39. Sperling P, Lee M, Girke T, Zahringer U, Stymne S, Heinz E acid biosynthesis in the protozoon Perkinsus marinus include a (2000) A bifunctional delta-fatty acyl acetylenase/desaturase plant-like FAE1 3-ketoacyl coenzyme A synthase. J Biol Chem from the moss Ceratodon purpureus. A new member of the 282:2996–3003 cytochrome b5 superfamily. Eur J Biochem 267:3801–3811 23. Reddy AS, Nuccio ML, Gross LM, Thomas TL (1993) Isolation 40. de Antueno RJ, Knickle LC, Smith H, Elliot ML, Allen SJ, of a delta 6-desaturase gene from the cyanobacterium Nwaka S, Winther MD (2001) Activity of human Delta5 and

123 Lipids

Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty {Delta}5-desaturase from Paramecium tetraurelia. Appl Envi- acids. FEBS Lett 509:77–80 ron Microbiol 77:1854–1861 41. Reddy AS, Thomas TL (1996) Expression of a cyanobacterial 56. Tonon T, Sayanova O, Michaelson LV, Qing R, Harvey D, delta 6-desaturase gene results in gamma-linolenic acid pro- Larson TR, Li Y, Napier JA, Graham IA (2005) Fatty acid duction in transgenic plants. Nat Biotechnol 14:639–642 desaturases from the microalga Thalassiosira pseudonana. 42. Truksa M, Vrinten P, Qiu X (2008) Metabolic engineering of FEBS J 272:3401–3412 plants for polyunsaturated fatty acid production. Mol Breed 57. Hastings N, Agaba M, Tocher DR, Leaver MJ, Dick JR, Sargent 23:1–11 JR, Teale AJ (2001) A vertebrate fatty acid desaturase with 43. Liu JW, Huang YS, DeMichele S, Bergana M, EJr Bobik, Delta 5 and Delta 6 activities. Proc Natl Acad Sci USA Hastilow C, Chuang LT, Mukerji P, Knutzon D (2001) Evalu- 98:14304–14309 ation of the seed oils from a canola plant genetically transformed 58. Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer to produce high levels of gamma-linolenic acid. In: Huang YS, U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain Ziboh VA (eds) Gamma-linolenic acid: recent advances in polyunsaturated fatty acids in transgenic oilseeds: constraints on biotechnology and clinical applications. AOCS Press, Cham- their accumulation. Plant Cell 16:2734–2748 paign, IL, pp 61–71 59. Cheng B, Wu G, Vrinten P, Falk K, Bauer J, Qiu X (2010) 44. Ursin VM (2003) Modification of plant lipids for human health: Towards the production of high levels of eicosapentaenoic acid development of functional land-based omega-3 fatty acids. in transgenic plants: the effects of different host species, genes J Nutr 133:4271–4274 and promoters. Transgenic Res 19:221–229 45. Eckert H, La VB, Schweiger BJ, Kinney AJ, Cahoon EB, 60. Voss A, Reinhart M, Sankarappa S, Sprecher H (1991) The Clemente T (2006) Co-expression of the borage Delta 6 desat- metabolism of 7,10,13,16,19-docosapentaenoic acid to urase and the Arabidopsis Delta 15 desaturase results in high 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent accumulation of stearidonic acid in the seeds of transgenic of a 4-desaturase J Biol Chem 266:19995–20000 soybean. Planta 224:1050–1057 61. Meyer A, Cirpus P, Ott C, Schlecker R, Zahringer U, Heinz E 46. Knauf VC, Shewmaker C, Flider F, Emlay D, Rey E (2011) (2003) Biosynthesis of docosahexaenoic acid in Euglena grac- Safflower with elevated gamma-linolenic acid. U.S. Patent ilis: biochemical and molecular evidence for the involvement of 7,893,321B2 a Delta4-fatty acyl group desaturase. Biochemistry 42:9779– 47. Knutzon DS, Thurmond JM, Huang YS, Chaudhary S, Bobik 9788 EG Jr, Chan GM, Kirchner SJ, Mukerji P (1998) Identification 62. Tripodi KE, Buttigliero LV, Altabe SG, Uttaro AD (2006) of Delta5-desaturase from Mortierella alpina by heterologous Functional characterization of front-end desaturases from try- expression in bakers’ yeast and canola. J Biol Chem 273: panosomatids depicts the first polyunsaturated fatty acid bio- 29360–29366 synthetic pathway from a parasitic protozoan. FEBS J 48. Michaelson LV, Lazarus CM, Griffiths G, Napier JA, Stobart 273:271–280 AK (1998) Isolation of a Delta5-fatty acid desaturase gene from 63. Tonon T, Harvey D, Larson TR, Graham IA (2003) Identifica- Mortierella alpina. J Biol Chem 273:19055–19059 tion of a very long chain polyunsaturated fatty acid Delta4- 49. Kajikawa M, Matsui K, Ochiai M, Tanaka Y, Kita Y, Ishimoto desaturase from the microalga Pavlova lutheri. FEBS Lett M, Kohzu Y, Shoji S, Yamato KT, Ohyama K, Fukuzawa H, 553:440–444 Kohchi T (2008) Production of arachidonic and eicosapentae- 64. Zhou XR, Robert SS, Petrie JR, Frampton DM, Mansour MP, noic acids in plants using bryophyte fatty acid Delta6-desatur- Blackburn SI, Nichols PD, Green AG, Singh SP (2007) Isolation ase, Delta6-elongase, and Delta5-desaturase genes. Biosci and characterization of genes from the marine microalga Pav- Biotechnol Biochem 72:435–444 lova salina encoding three front-end desaturases involved in 50. Kajikawa M, Yamato KT, Kohzu Y, Nojiri M, Sakuradani E, docosahexaenoic acid biosynthesis. Phytochemistry 68:785–796 Shimizu S, Sakai Y, Fukuzawa H, Ohyama K (2004) Isolation 65. Li Y, Monroig O, Zhang L, Wang S, Zheng X, Dick JR, You C, and characterization of delta(6)-desaturase, an ELO-like enzyme Tocher DR (2010) Vertebrate fatty acyl desaturase with Delta4 and delta(5)-desaturase from the liverwort Marchantia poly- activity. Proc Natl Acad Sci USA 107:16840–16845 morpha and production of arachidonic and eicosapentaenoic 66. Kinney AJ, Cahoon EB, Damude HG, Hitz WD, Kolar CW, Liu acids in the methylotrophic yeast Pichia pastoris. Plant Mol Biol ZB (2004) Production of very long chain polyunsaturated fatty 54:335–352 acids in oilseed plants. WO 2004/071467 51. Saito T, Ochiai H (1999) Identification of Delta5-fatty acid 67. Wu G, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, desaturase from the cellular slime mold Dictyostelium discoid- Heinz E, Qiu X (2005) Stepwise engineering to produce high eum. Eur J Biochem 265:809–814 yields of very long-chain polyunsaturated fatty acids in plants. 52. Cho HP, Nakamura M, Clarke SD (1999) Cloning, expression, Nat Biotechnol 23:1013–1017 and fatty acid regulation of the human delta-5 desaturase. J Biol 68. Robert SS, Singh SP, Zhou X, Petrie JR, Blackburn SI, Mansour Chem 274:37335–37339 PM, Nichols PD, Liu Q, Green AG (2005) Metabolic engi- 53. Hong H, Datla N, MacKenzie SL, Qiu X (2002) Isolation and neering of Arabidopsis to produce nutritionally important DHA characterization of a delta5 FA desaturase from Pythium irreg- in seed oil. Funct Plant Biol 32:473–479 ulare by heterologous expression in Saccharomyces cerevisiae 69. Qi B, Fraser TC, Bleakley CL, Shaw EM, Stobart AK, Lazarus and oilseed crops. Lipids 37:863–868 CM (2003) The variant ‘his-box’ of the C18-Delta9-PUFA- 54. Kaewsuwan S, Cahoon EB, Perroud PF, Wiwat C, Panvisavas specific elongase IgASE1 from Isochrysis galbana is essential N, Quatrano RS, Cove DJ, Bunyapraphatsara N (2006) Identi- for optimum enzyme activity. FEBS Lett 547:137–139 fication and functional characterization of the moss Physcomit- 70. Park WJ, Kothapalli KS, Lawrence P, Tyburczy C, Brenna JT rella patens delta5-desaturase gene involved in arachidonic and (2009) An alternate pathway to long-chain polyunsaturates: the eicosapentaenoic acid biosynthesis. J Biol Chem 281:21988– FADS2 gene product Delta8-desaturates 20:2n–6 and 20:3n–3. 21997 J Lipid Res 50:1195–1202 55. Tavares S, Grotkjaer T, Obsen T, Haslam RP, Napier JA, 71. Sayanova O, Haslam RP, Caleron MV, Lopez NR, Worthy C, Gunnarsson N (2011) Metabolic engineering of Saccharomyces Rooks P, Allen MJ, Napier JA (2011) Identification and func- cerevisiae for production of eicosapentaenoic acid, using a novel tional characterisation of genes encoding the omega-3

123 Lipids

polyunsaturated fatty acid biosynthetic pathway from the coc- residues adjacent to histidine-rich motif II. Biochem Biophys colithophore Emiliania huxleyi. Phytochemistry 72:594–600 Res Commun 339:1029–1034 72. Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, 86. Libisch B, Michaelson LV, Lewis MJ, Shewry PR, Napier JA Napier JA, Stobart AK, Lazarus CM (2004) Production of very (2000) Chimeras of Delta6-fatty acid and Delta8-sphingolipid long chain polyunsaturated omega-3 and omega-6 fatty acids in desaturases. Biochem Biophys Res Commun 279:779–785 plants. Nat Biotechnol 22:739–745 87. Lange SJ, Que L Jr (1998) Oxygen activating nonheme iron 73. Petrie JR, Shrestha P, Belide S, Mansour MP, Liu Q, Horne J, enzymes. Curr Opin Chem Biol 2:159–172 Nichols PD, Singh SP (2011) Transgenic production of arachi- 88. Shanklin J, Guy JE, Mishra G, Lindqvist Y (2009) Desaturases: donic acid in oilseeds. Transgenic Res. doi:10.1007/s11248- emerging models for understanding functional diversification of 011-9517-7 diiron-containing enzymes. J Biol Chem 284:18559–18563 74. Shanklin J, Somerville C (1991) Stearoyl-acyl-carrier-protein 89. Whyte LG, Schultz A, Beilen JB, Luz AP, Pellizari V, Labbe D, desaturase from higher plants is structurally unrelated to the Greer CW (2002) Prevalence of alkane monooxygenase genes in animal and fungal homologs. Proc Natl Acad Sci USA Arctic and Antarctic hydrocarbon-contaminated and pristine 88:2510–2514 soils. FEMS Microbiol Ecol 41:141–150 75. Stymne S, Stobart AK (1986) Biosynthesis of gamma-linolenic 90. Feussner I, Wasternack C, Kindl H, Kuhn H (1995) Lipoxyge- acid in cotyledons and microsomal preparations of the devel- nase-catalyzed oxygenation of storage lipids is implicated in oping seeds of common borage (Borago officinalis). Biochem J lipid mobilization during germination. Proc Natl Acad Sci USA 240:385–393 92:11849–11853 76. Griffiths G, Stobart AK, Stymne S (1988) Delta 6- and delta 91. Arciero DM, Orville AM, Lipscomb JD (1985) [17O]Water and 12-desaturase activities and phosphatidic acid formation in nitric oxide binding by protocatechuate 4,5-dioxygenase and microsomal preparations from the developing cotyledons of catechol 2,3-dioxygenase. Evidence for binding of exogenous common borage (Borago officinalis). Biochem J 252:641–647 ligands to the active site Fe2? of extradiol dioxygenases. J Biol 77. Domergue F, Abbadi A, Ott C, Zank TK, Zahringer U, Heinz E Chem 260:14035–14044 (2003) Acyl carriers used as substrates by the desaturases and 92. Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are elongases involved in very long-chain polyunsaturated fatty catalytically essential in a membrane-associated iron enzyme, acids biosynthesis reconstituted in yeast. J Biol Chem stearoyl-CoA desaturase, and are conserved in alkane hydroxylase 278:35115–35126 and xylene monooxygenase. Biochemistry 33:12787–12794 78. Petrie JR, Shrestha P, Mansour MP, Nichols PD, Liu Q, Singh 93. Fauconnot L, Buist PH (2001) Gamma-linolenic acid biosyn- SP (2010) Metabolic engineering of omega-3 long-chain poly- thesis: cryptoregiochemistry of delta6 desaturation. J Org Chem unsaturated fatty acids in plants using an acyl-CoA Delta6- 66:1210–1215 desaturase with omega3-preference from the marine microalga 94. Behrouzian B, Buist PH (2003) Mechanism of fatty acid Micromonas pusilla. Metab Eng 12:233–240 desaturation: a bioorganic perspective. Prostaglandins Leukot 79. Sperling P, Ternes P, Zank TK, Heinz E (2003) The evolution of Essent Fatty Acids 68:107–112 desaturases. Prostaglandins Leukot Essent Fatty Acids 68:73–95 95. Meesapyodsuk D, Reed DW, Savile CK, Buist PH, Schafer UA, 80. Stukey JE, McDonough VM, Martin CE (1989) Isolation and Ambrose SJ, Covello PS (2000) Substrate specificity, regiose- characterization of OLE1, a gene affecting fatty acid desatura- lectivity and cryptoregiochemistry of plant and animal omega-3 tion from Saccharomyces cerevisiae. J Biol Chem 264:16537– fatty acid desaturases. Biochem Soc Trans 28:632–635 16544 96. Aguilar PS, de MD (2006) Control of fatty acid desaturation: a 81. Mitchell AG, Martin CE (1995) A novel cytochrome b5-like mechanism conserved from bacteria to humans. Mol Microbiol domain is linked to the carboxyl terminus of the Saccharomyces 62:1507–1514 cerevisiae delta-9 fatty acid desaturase. J Biol Chem 270: 97. Napier JA (2007) The production of unusual fatty acids in 29766–29772 transgenic plants. Annu Rev Plant Biol 58:295–319 82. Qiu X, Hong H, Datla N, MacKenzie SL, Taylor DC, Thomas 98. Sayanova O, Napier JA (2011) Transgenic oilseed crops as an TL (2002) Expression of borage delta-6 desaturase in Saccha- alternative to fish oils. Prostaglandins Leukot Essent Fatty Acids romyces cerevisiae and oilseed crops. Can J Bot 80:42–49 85:253–260 83. Hongsthong A, Subudhi S, Sirijuntarut M, Kurdrid P, Cheevadh- 99. Singh SP, Zhou XR, Liu Q, Stymne S, Green AG (2005) Met- anarak S, Tanticharoen M (2006) Revealing the complementation abolic engineering of new fatty acids in plants. Curr Opin Plant of ferredoxin by cytochrome b (5) in the Spirulina-(6)-desaturation Biol 8:197–203 reaction by N-terminal fusion and co-expression of the fungal- 100. Napier JA, Graham IA (2010) Tailoring plant lipid composition: cytochrome b (5) domain and Spirulina-(6)-acyl-lipid desaturase. designer oilseeds come of age. Curr Opin Plant Biol 13:330–337 Appl Microbiol Biotechnol 72:1192–1201 101. Graham IA, Larson T, Napier JA (2007) Rational metabolic 84. Sayanova O, Shewry PR, Napier JA (1999) Histidine-41 of the engineering of transgenic plants for biosynthesis of omega-3 cytochrome b5 domain of the borage delta6 fatty acid desaturase polyunsaturates. Curr Opin Biotechnol 18:142–147 is essential for enzyme activity. Plant Physiol 121:641–646 102. Cahoon EB, Shockey JM, Dietrich CR, Gidda SK, Mullen RT, 85. Na-Ranong S, Laoteng K, Kittakoop P, Tanticharoen M, Dyer JM (2007) Engineering oilseeds for sustainable production Cheevadhanarak S (2006) Targeted mutagenesis of a fatty acid of industrial and nutritional feedstocks: solving bottlenecks in Delta6-desaturase from Mucor rouxii: role of amino acid fatty acid flux. Curr Opin Plant Biol 10:236–244

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