Introduction

Fatty acid desaturases (FADs) are a group of enzymes that catalyze desaturation reaction by introducing double bonds in a wide range of different positions and desaturating saturated fatty acids into unsaturated fatty acids. This reaction takes place in the presence of molecular oxygen and reducing equivalents (which are delivered by an electron transport system) (Los and Murata 1998). The FADs have an Enzyme Commission (EC) number of 1.14.19.X and have been classified as oxidoreductases that act on single donors by incorporating with molecular oxygen (oxygenase), usually by utilizing NADP or NAD + as co-factors. The distribution of FADs is nearly universal with a few exceptions, such as Escherichia coli (Los and Murata 1998). The FADs can aid in the desaturation of fatty acids, which results in monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). In the production of PUFAs, FADs are essential; PUFAs serve as precursors of eicosanoids, pheromones, growth regulators, and hormones, among other biologically active compounds (Los and Murata 1998). The biosynthesis of PUFAs can occur via different pathways, and they benefit in the development of medicines and nutritional supplements (Lindqvist et al. 1996). Fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (found primarily in seafood sources, fish-oil supplements, seeds, and nuts), are the end products of desaturation and elongation processes, and are essential for human health and the physiology of the human body as they are a nutritional necessity. PUFAs are produced in large quantities by fungi, algae, and protozoa, and other microbes. These microorganisms are expected to have all the enzymes required to produce EPA and DHA (Pereira et al. 2003).

Recent findings on the challenges and strategies for protein production enabled the determination of membrane protein structural biology, making it possible for the membrane protein to be solved. The advancement of technology has made the crystallization and structural elucidation of membrane-bound desaturase as one of the most significant achievements of the recent years. The number of solved crystal structures of FADs has increased tremendously. X-ray structure of a Δ9 mouse stearoyl-CoA desaturase has been crystallized and solved at 2.6 Å resolution by Bai et al. (2015). Another crystal structure of Δ9 in human stearoyl-coenzyme A desaturase in complex with substrate has been elucidated (Wang et al. 2015a, b). Both structures exhibit a tertiary fold that resembles a mushroom, with the catalytic cap domain and terminal ends on the cytosolic side of the membrane (Nachtschatt et al. 2020). Mouse and human Δ9 FAD crystal structure with the di-iron center were studied; however, in the catalytic site of both proteins, zinc ions were found instead of iron ions, which thereby triggered the enzymes to become inactive (Bai et al. 2015; Wang et al. 2015a, b). In this manuscript, the biosynthesis pathway, catalytic mechanism, architecture, and roles of unsaturated FADS are discussed.

Classification of FADs

Two groups of FADs have been found in nature: soluble and membrane-bound desaturases. These groups have been broadly classified into two evolutionary unrelated groups of soluble acyl-acyl carrier protein (ACP) and membrane-bound desaturases (Lou et al. 2014). Soluble desaturase is present in the plastids of higher plants that require NADPH and oxygen. It is part of an electron transport chain that includes ferredoxin-NADPH reductase. Each acyl-ACP desaturase forms a reactive complex with oxygen by binding two iron atoms (Shanklin and Cahoon 1998). The conserved motif for soluble FADs is 2xD/EX2H (Shanklin and Cahoon 1998). On the other hand, membrane FADs is found in most prokaryotes and eukaryotic organisms. The membrane-bound group is further subdivided into acyl-lipid FADs e and acyl-coenzyme A (CoA) FADs. Membrane FADs use ferredoxin or cytochrome b5. In monogalactosyl diacylglycerol, cyanobacterial membrane FADs could desaturate stearic (18:0) and oleic (18:1 n-9) fatty acyl chain (Tocher et al. 1998). Most of this type of FADs has 300–350 amino acid residues, and it is a hydrophobic protein that spans membranes four times (Murata and Wada 1995). The endoplasmic reticulum membranes of animals, yeasts, and fungi contain acyl-CoA FADs, and they obtain electrons from cytochrome b5 and NADH-dependent cytochrome b5 reductase. All mammalian FADs that have been identified are grouped under acyl-CoA desaturases (Tocher et al. 1998).

The amino acid sequences of membrane-bound FADs suggest that these enzymes contain two long hydrophobic domains that can traverse the membrane bi-layer twice (Alberts 2002). Three conserved His-box motifs were discovered through sequence comparison: HX3-4H, HX2-3HH, and H/QX2-3HH (H-Histidine; X-variable amino acid; Q-glutamine), all of which include eight histidine residues. These histidine residues operate in the catalytic center of desaturases as possible ligands for iron atoms (Nakamura and Nara 2004). These iron atoms are defined as nonheme and di-iron containing; thus, they can bind the iron ions with conserved histidine residues. All known desaturases have been characterized by the presence of three histidine clusters, which are strongly conserved in the amino acid sequence (Wang et al. 2013). Table 1 shows the comparison of two types of fatty acid desaturases. According to Nachtschatt et al. (2020), linear saturated fatty acids have been created by the fatty acid synthase enzyme system known as FAS I or FAS II, which has the potential to elongate acetyl CoA by condensing it with malonyl-CoA molecules under the presence of carbon dioxide and water in cyclical manner. Even-numbered carbons of fatty acids are the most prevalent, and palmitic acids along with stearic acid are the most prevalent products of fatty acid synthesis (Li et al. 2016a, b, c). Membrane-bound desaturases can be later further distinguished by the position of fatty acid where the double bond has been introduced (Nachtschattt et al. 2020).

Table 1 Comparison of features of soluble and membrane-bound desaturases. Sources from Nakamura and Nara (2004), Shanklin et al. (2009), and Wang et al. (2013)
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Desaturases can be further classified as first desaturases, methyl-end desaturases, and front-end desaturases, based on the position of the introduced double bond (Li et al. 2016a, b, c). First desaturase introduces first double bond into the saturated acyl chain, and the predominant member is stearoyl-coenzyme A desaturase which generally introduces a double bond at the 9th position of palmitic acid (C16:0) or stearic acid (C18:0). Front-end desaturases such as Δ4, Δ5, and Δ6 can introduce double bonds between existing double bonds and the carboxyl group (-COOH) to generate PUFAs (Lee et al. 2016). According to International Union of Pure and Applied Chemistry (IUPAC) in the Compendium of Chemical Terminology (Castro et al. 2016), an enzyme from this group is referred to as Δx (delta X desaturase), where X is the number corresponding to the position from the carboxyl end at which the double bond is inserted. Desaturases with a new double bond between an existing unsaturation and the PUFA’s methyl terminus (–CH3) are known as methyl-end desaturases and referred to as ωx (omega X desaturases) (Monroig and Kabeya 2018). Therefore, a double bond at 12th position of fatty acyl chain counted from the carboxyl-end is known as Δ12 desaturase, and ω6 when counted from the methyl-end.

Few membrane-bound desaturases have been reported from organisms, such as Δ9, Δ12, Δ15, Δ6, Δ5, and Δ4. Δ9 FAD genes catalyze the insertion of double bond at the position of the 9th carbon in fatty acids, synthesizing long-chain monounsaturated fatty acids from long-chain saturated fatty acids, which convert stearic acid (C18:0) and palmitic acid (C16:0) to oleic acid (OA, C18:1Δ9) and palmitoleic acid (POA, C16:1Δ9) (Zhuang et al. 2022). It is commonly known as Stearoyl CoA, and has been isolated from numerous species, such as Saccharomyces cerevisiae, Mortierella alpina, Histoplasma capsulatum, Tetrahymena thermophile (Tocher et al. 1998; Sakuradani et al. 1999; Laoteng et al. 1999), and Pseudomonas oleovorans (Li et al. 2009). Two hydrophobic domains of this desaturase can penetrate the phospholipid bilayer (Zhang 2009). Formed from red algae (known as Cyanidioschyzon merolae) and yeast, Δ9 FADs are distinguished by the existence of a cytochrome b5 domain located at the C-terminus, which is obligatory for the protein’s activity (Mitchell and Martin 1995; Itoh et al. 1998). Δ9 FADs has been reported in humans (Wang et al. 2015a, b) and mice (Bai et al. 2015). Extensive studies of this protein have been conducted in these two organisms up until structure elucidation (Wang et al. 2015a, b and Bai et al. 2015).

Δ12 FADs catalyze the insertion of a double bond at the position of the 12th carbon in fatty acids, indicating the double bonds are placed at the position of the 6th carbon position counted from methyl end groups. It is a vital enzyme catalyzing oleic acid (OA, C18:1Δ9) to linoleic acid (LA, C18:2Δ9,12) (Lee et al., 2016) (Saini et al. 2021). Δ12 FADs have been isolated from Thraustochytrium aureum (yeasts) (Matsuda et al. 2012) and characterized from Mortierella alpina (Huang et al. 1999) and Mucor rouxii (Passorn et al. 1999). A study suggested that Δ12 FADs found in Mortierella alpina are extremely active and cause a conversion of 71.4% of substrate oleic acid to linoleic acid. In Lipomyces starkeyi yeast, the activity of Δ12 FADs has been measured (Lomascolo et al. 1996); in microorganisms, Δ12 FADs are quite similar to such genes in animals in terms of sequence identity. According to Zhuang et al. (2022), identification and characterization of Δ12 FADs genes from Isochrysis galbana (Han et al. 2019), Acanthamoeba castellani (Sayanova et al. 2016), Chlamydomonas sp. (Zhang et al. 2011), Calendula officinalis (Qiu et al. 2001), Helianthus annuus (Martinez-Rivas et al. 2001), Chlorella vulgaris (Lu et al. 2009), Phaedactylum tricornutum (Domergue et al. 2003), and Haematococcus pluvialis (Zhang et al. 2020) showed they shared three conserved histidine box motifs, which known as catalytic center and are critical for desaturase activity.

Δ15 FADs, also known as ω3 (counted from methyl end), add a double bond at the 15th position in fatty acids, indicating the double bonds at the 3rd position of fatty acids from methyl end groups. Δ15 FADs catalyze linoleic acid (LA, C18: 2Δ9,12) and γ-linolenic acid (GLA, C18: 3Δ6,9,12) to produce α-linolenic acid (ALA, C18: 3Δ9,12,15) and stearidonic acid (SDA, C18: 4Δ6,9,12,15), respectively (Saini et al. 2021). Δ15 FADs have been discovered in various species of molds, such as Saprolegnia diclina (Pereira et al. 2004), Phytophthora infestans (Fu et al. 2013), Pythium aphanidermatum (Xue et al. 2013), and blue-green algae, Synechocystis sp. PCC6803 (Drew et al. 2001). A previous study showed that Δ15 desaturase of Phytophthora infestans converted 30.94% of arachidonic acid (AA) into eicosapentaenoic acid (EPA) (Fu et al. 2013). Δ12 and Δ15 FADs are important enzymes in the synthesis of omega-3 and omega-6 in mammals, as mammals cannot produce these fatty acids. According to phylogenetic studie, Δ12 FADs is an ancestor gene of Δ15 FADs (Wang et al. 2013).

Δ6 FADs add a double bond at the 6th carbon–carbon bond position from the carboxylic acid end in fatty acids. The desaturases are a rate-limiting enzyme in generating γ-linolenic acid (GLA, C18:3Δ6,9,12) and producing stearidonic acid (SDA, C18:4Δ6,9,12,15) from linoleic acid (LA, C18:2Δ9,12) and α-linolenic acid (ALA, C18:3Δ9,12,15) in the synthesis of PUFAs in mammals and humans (Zhuang et al. 2022). These membrane-bound FADs have been identified in Synechocystis sp. PCC6803, Mortierella alpina (Chen et al. 2014), and Mucor rouxii (Laoten et al. 2000). However, Δ6 FADs of Mucor rouxii differ from those of Mortierella alpina as it has around 500 amino acid sequences and the identity of the sequence is closely related to plant Δ6 FADs compared to animal or fungal Δ6 FADs. It also has unique histidine-conserved motifs of “HKHHSH,” which cannot be found in any other desaturase. An early study stated that the enzymatic activity is thought to be dependent on the histidine-conserved region (Laoten et al. 2000).

Δ5 FADs catalyze the addition of a double bond at the 5th carbon–carbon bond from the carboxylic acid end in fatty acids. They catalyze the conversion of dihomo-γ-linolenic acid (DGLA, C20:3Δ8,11,14) and eicosatetraenoic acid (ETA, C20:4Δ8,11,14,17) to arachidonic acid (AA, C20:4Δ5,8,11,14) and eicosapentaenoic acid (EPA, C20:4Δ5,8,11,14,17) (Lee et al. 2016). These genes have been determined first in a fungus, Mortierella alpina, and the heterokont algae, Thraustochytrium sp. (Michaelson et al. 1998). According to Hastings et al. (2001) and Wang et al. (2014), Δ5 FADs in marine invertebrates showed bifunctional activity with the desaturation reaction of Δ5 and Δ6 FADs. Δ5 FADs have also been functionally expressed in yeast. A study showed that two types of Δ5 FADs have been identified in slime mold, Dictyostelium discoideum, and they shared 66% similarities, and their protein predictions had a sequence similarity of 38.6–42% with Mortierella alpina’s Δ5 FADs (Saito et al. 2000). The amino acid sequence from these FADs contained three conserved histidine box motifs and was significantly homologous (Zhuang et al. 2022).

Biosynthesis of FADs

The biosynthesis of unsaturated fatty acids is synthesized de novo from acetyl-coenzyme (CoA) as a source of carbon, especially the C16:0 and C18:0 in animals (Garba et al. 2017). The pathway is catalyzed by the multienzyme fatty acid synthase (FAS) complex and acetyl-CoA carboxylase. The FAS complex is a key enzymatic complex in fatty acid synthesis from acetyl-CoA and malonyl-CoA in the presence of NADPH (Beld et al. 2015). The complex can be broadly divided into two types, Type 1 system (mainly found in animals and yeast, uses a single large multifunctional polypeptide) and the Type II system (mostly found in prokaryotes and plants, uses a series of monofunctional enzymes) (Garba et al. 2017). A protein called the acyl carrier protein (ACP) is required by both type I and type II FAS to transport the fatty acid from enzyme to enzyme (Beld et al. 2015). ACPs move and present the expanding acyl chain to the proper reaction partners for elongation and the synthesis of fatty acids.

The pathway was first started with two acetyl-CoA, one converted to malonyl-CoA by the enzyme acetyl-CoA carboxylase (Ahern and Rajagopal 2021). This enzyme is known to be phosphorylated by both AMP Kinase and Protein Kinase A. Both molecules have their CoA portions replaced by acyl-carrier protein (ACP) to form acetyl-ACP and malonyl-ACP (Ahern and Rajagopal 2021). Then, the chemical reactions resemble those of beta oxidation reversed. The ketone is reduced to a hydroxyl using NADPH, and water is removed from carbons 2 and 3 of the hydroxyl intermediate to produce a trans-doubled bonded molecule after every round of elongation. Last, the double bond is hydrogenated to yield a saturated intermediate via a chronological action by ketoactyl reductase, dehydrase, and enoyl reductase activities (Garba et al. 2017). Elongation of fatty acids to make fatty acids longer than 16 carbons is catalyzed by elongase enzymes and occurs in the endoplasmic reticulum. Fatty acids can also be extended by mitochondria, but their starting materials are typically less than 16 carbons long. Both environments use similar methods to the cytoplasm. Desaturation happens after fatty acids are produced in their saturated state (Ahern and Rajagopal 2021). Desaturases are enzymes that facilitate the cis-double bond synthesis in mature fatty acids.

Mechanism of FADS

The desaturation reaction catalyzed by fatty acid desaturase is an aerobic process (Czumaj and Sledzinski 2020). PUFAs are synthesized through chloroplasts (prokaryotic) and endoplasmic reticulum through ER (eukaryotic) pathways (He et al. 2020). The reaction requires oxygen, cytochrome b5, and NADH-dependent cytochrome b5 reductase, whereby oxygen acts as a hydrogen acceptor, and cytochrome b5 as an electron carrier (Shanklin et al. 2009). NADPH is considered essential for hydrogen donors for the synthesis of < 18C fatty acids (Chen et al. 2013). As in eukaryotic cells, acyl chains require to be the first ones to be exported to the ER (LaBrant et al. 2018; Li et al. 2016a, b, c). At the initial stage of desaturation reaction, reduced cytochrome b5 interacts with the non-heme iron active site of the desaturase, which enables the enzyme to react with oxygen (O2) and substrate, and results in the activation of hydrogen at the carbon of incipient double bond in substrate, producing a very short-lived carbon-centered radical as the product (Martin-Montalvo et al. 2016; Zhang et al. 2016). The next stage of this reaction shows that the molecule loses the second hydrogen via rapid disproportionation, which leads to the production of the final product with the formation of a new double bond, along with two molecules of water (Behrouzian and Buist 2003). Saturated fatty acids (SFAs), such as stearic acid (C18:0) and palmitic acid (C16:0), can be unsaturated by all eukaryotes and most aerobic bacteria, whereas the majority of prokaryotic organisms only contain of the so-called anaerobic fatty acid synthesis pathway that does not include any desaturase at all (Keweloh and Heipieper 1996).

Role of unsaturated FADs

Unsaturated FADs play a crucial role in the synthesis of PUFAs, inserting additional unsaturated bonds into the acyl chain producing products of these enzymes. The organism in which desaturases are expressed determines their exact function. FADs seem to exert a variable effect on microorganisms, plants, humans, and other mammals.

Role of unsaturated FADs in microorganisms

In bacteria, changes in the PUFAs composition in the plasma membrane could be vital in the adjustment towards harsh environmental conditions; thus, microorganisms can change the activity of desaturase to adapt PUFAs content towards temperature, pH, and atmospheric pressure to maintain appropriate fluidity of plasma membranes as the melting temperature of PUFAs is much lower than saturated and monounsaturated fatty acids (Yoshida et al. 2016). According to Hazel and Williams (1990), conditions of low temperature and high pressure reveal the integrity of membranes, which can be irreversible, from a fluid to a rigid state. PUFAs in microorganisms, usually with a long chain of fatty acids (around 16–18 carbons), have been found in great amounts in cold-adapted bacteria within the polar regions and deep-sea areas. Therefore, PUFAs especially EPA and DHA have been assumed as effective modulators for modifying membrane fluidity (Yoshida et al. 2016). The introduction of these bacteria into a warmer environment resulted in changes in expression of desaturases as well as changes in the PUFAs content in the plasma membrane (Diomandé et al. 2015; Yoshida et al. 2016; Siliakus et al. 2017; Bale et al. 2019).

Role of unsaturated FADs in plants

Plant composition of fatty acids is crucial for the growth and vegetation of plants as desaturation of fatty acids influences the tolerance of plants towards environmental stressors (Shanklin and Cahoon 1998; Iba 2002). According to Venegas-Caleron et al. (2009), PUFAs with more than 20 carbons cannot often be produced by higher plants, except for a few species that produce GLA and stearidonic acid. However, the production of transgenic plant oil has been developed and the first transgenic plant reported was tobacco, which used cyanobacterial delta-6 desaturase to accumulate GLA (Reddy and Thomas 1996). In plants, changes in fatty acid desaturase play roles in the facilitation of cold adaptation, maintenance of normal fluidity, and integrity of the plasma membrane (Iba 2002). A study revealed that the presence of fatty acid desaturases, FAD2 and FAD6 in seedlings, showed a significant impact on the activation by salt and osmotic stress (Zhang et al. 2009), while the deficiency of FAD2 resulted in the accumulation of sodium ions in the cytoplasm of root cells and a rise in sensitivity towards salt stress during germination and early seedling growth (Zhang et al. 2012).

Role of unsaturated FADs in human and mammals

FADs appear to have a varied effect on human health, which is the outcome of an association among polymorphisms of desaturase towards certain metabotypes (Wang et al. 2014). It is well-recognized that among these fatty acids, ω3 and ω6 are the most important, as the biosynthesis of these two fatty acids creates health benefits throughout life. r ω3 and ω6 fatty acids are crucial in humans, as they cannot be produced de novo. Research suggested that the body could convert ALA to DHA and EPA throughout the desaturation and elongation processes; however, the conversion process produces a small number of fatty acids (Chiu et al. 2008), around 0.3% for EPA and < 0.01% for DHA (Hussein et al. 2005). Low levels of EPA and DHA are correlated with various inflammatory processes, including cardiovascular diseases, poor fetal development (Swanson et al. 2012), Alzheimer’s diseases (Swanson et al. 2012), bipolar affective disorders, schizophrenia (Liu et al. 2011; Li et al. 2009; Yao et al. 2000), and cardiovascular disease (Swanson et al. 2012). In a study that used human blood samples conducted by Bouwens et al. (2009), they mentioned the intake of EPA and DHA changed the expression of 1040 genes and resulted in a decrease in the expression of genes associated with inflammatory and atherogenesis-related pathways, such as adipogenesis and hypoxia signaling. Schiano et al. (2008) discovered that EPA and DHA supplementation improved endothelial function in patients with atherosclerosis by lowering plasma levels of soluble thrombomodulin from a median value of 33.0 to 17.0 mg/L (P = 0.04) and increasing brachial artery flow–mediated dilation from 6.7 to 10.0% (P = 0.02). Therefore, it is important to take diets with a higher content of EPA and DHA, which include seafood sources, such as fish and dietary supplements derived from fish and fish oil (Leaf and Hatcher 2009; Mann et al. 2010; Saito et al. 2008).

The beneficial effect of DHA has also been recognized in various types of chronic inflammatory conditions, as DHA can be metabolized into anti-inflammatory bioactive lipid mediators (Nagy and Tiuca 2017). These characteristics have been conservatively defined as their ability to interact with primary inflammatory signaling pathways and their inhibition of inflammatory cytokine production (Lorente-Cebrian et al. 2015). A report described that higher concentration of DHA is associated with fewer morning stiffness, swelling joints, and pain. Collagen-induced arthritis is less common and severe when these fatty acids are consumed (Nagy and Tiuca 2017). DHA may also inhibit the production of reactive oxygen species and the stimulation of TNFRI (tumor necrosis factor receptor type I) by AA, both of which have immune-modulatory qualities and can affect T and B cell activity (Brouwers et al. 2015). Through the nuclear peroxisome proliferator–activated receptor (PPAR) system, DHA and EPA may suppress the expression of genes linked to inflammation (Endo and Arita 2016). This receptor can activate and stimulate β-oxidation, lowering the levels of triglycerides and fatty acids in the bloodstream, which are responsible for the prevention of hyperplasia and adipocyte hypertrophy (Echeverria et al. 2016). A study reported that patients with rheumatoid arthritis have a low level of DHA, EPA, and some fatty acids (such as oleic acid and palmitic acid) in comparison to normal healthy controls (Brouwers et al. 2015). In addition, some MUFAs produced by the desaturation reaction have a beneficial effect on anti-inflammatory properties, such as oleic acid (Zhou et al. 2016). According to a recent study, taking a higher amount of DHA lowers the amount of AA in inflammatory cell membranes, which lowers the amount of pro-inflammatory eicosanoids (Nagy and Tiuca 2017).

As DHA is abundant in brain cells and retina for normal cell membrane function, FADs play a crucial role in proper fetal development and healthy aging (Dunstan et al. 2007; Krauss-Etschmann et al. 2007). AA, EPA, and DHA play a significant role in the growth of infants, and the development of their neurons and immune systems (Mychaleckyi et al. 2018; Powell et al. 2016; Xie et al. 2008; Muc et al. 2015; Ding et al. 2016). The key ingredients of the neuronal membranes are derived from AA and DHA (Czumaj and Sledzinski 2020). The presence of EPA and DHA in membrane phospholipids may affect a variety of biological mechanisms and pathways in the brain, including the integrity and survival of neurons, glial cells, endothelial cells, neurotransmission (dopaminergic, serotonergic, glutamatergic, and cholinergic), and neuroinflammation (Nishida and Murata 1996; Healy-Stoffel and Levant 2018; Cao et al. 2018). A study suggested that EPA and DHA are the key components associated with multiple benefits in an early stage of life (Swanson et al. 2012). As the placenta provides nutrients to the fetus, a certain amount of DHA is also transferred (Helland et al. 2008). Supplementing with EPA and DHA during pregnancy has been linked to longer gestation and higher fatty acid concentrations in fetal tissues, which may reduce the risk of preterm delivery by lowering prostaglandin E2 and prostaglandin F2a synthesis (Olsen et al. 2008; Roman et al. 2006). There is also evidence that EPA and DHA supplementation during pregnancy and lactation may protect infants against allergies, as fish oil supplementation generates a lower level of body cells linked to inflammation and immunological response (Krauss-Etschmann et al. 2008). Fatty acids are delivered to newborns through nursing, which contributes to the development of cognitive function and intellectual development (Capsi et al. 2007; Gould and Smithers 2019).

Though both ω3 and ω6 fatty acids are considered to have beneficial health effects, overconsumption of ω6 fatty acids with low intake of ω3 in modern westernized diet styles has been shown to be linked to antagonistic metabolic functions and the emergence of many chronic diseases, including inflammatory diseases, cardiovascular diseases, and some cancers related to inflammation (Mariamenatu and Abdu 2021). Large amounts of LA and AA derivatives cause cell proliferation, the endocannabinoid system to be extremely hyperactive, thrombus and atheroma formation, allergy, and inflammatory problems, especially in vulnerable individuals (Mariamenatu and Abdu 2012), even though these two PUFAs are essential as the human body is genetically incapable of synthesising them (Ruiz-Lopez et al. 2012). It was once thought that our ancestors consumed ω6 and ω3 in a ratio of 1–2/1, which is believed to be a perfect and balanced ratio during the Paleolithic period (a time of evolution) (Simopoulos 2008). To date, the current estimate for the typical ratio in western diet types is 20/1 (Sokola-Wysoczanska et al. 2018; Simopoulos 2016), whereas in South Asia, the ratio can reach 50/1 (Chaves et al. 2019). Increase in the ω3 and ω6 fatty acids ratio increases the risk of overweight and obesity (Simopoulos 2016). According to Simopoulos (2010), keeping the ratio of ω3 and ω6 fatty acids in balance helps lower the chance of developing autoimmune and possibly neurodegenerative disorders, as well as coronary heart disease, high blood pressure, cancer, type 2 diabetes, and arthritis.

Architecture of FADs

To date, there are around 59 crystal structures of FADs, and most of them are soluble desaturase, including the X-ray structure of Mycobacterium tuberculosis H37Rv DesA2 (Dyer et al. 2005), a soluble acyl-ACP desaturase (Buist 2004) and crystal structure of multifunctional Δ4 acyl-ACP desaturase isolated from English ivy (Hedera helix) plant at 1.95 Å resolution (Guy et al. 2007). Few membrane-bound FAD structures have been solved, which include X-ray structure of mouse stearoyl-CoA desaturase (Δ9 mSCD1) at 2.6 Å resolution (Bai et al. 2015), crystal structure of human stearoyl–coenzyme A (Δ9 hSCD1) desaturase at resolution 3.25 Å (Wang et al. 2015a, b), and Δ9 acyl-CoA desaturase from Mus musculus at 3.51 Å resolution (Shen et al. 2020). As of today, none of the crystal structures of membrane-bound FADs from bacteria have been established and solved; however, the structure prediction of Δ9 acyl-CoA FADs from Pseudomonas sp. AMS8 has been predicted (Garba et al. 2018) (shown in Table 2). It remains a major problem in determining crystal structures due to factors such as difficulties to express and purify (Birch et al. 2018), and identification of suitable conditions that would produce crystals for structure determination. Affinity chromatography is by far the most useful and widely used technology for purifying integral membrane proteins as done by these studies (Chen et al. 2013; Wang et al. 2017; Halim et al. 2021). Recombinant FADs of Δ9, Δ12, and Δ15 from Mortierella alpina were successfully affinity purified on His Mag Sepharose Ni beads with one-step purification in methylotrophic yeast, Pischia pastoris (Chen et al. 2013). Based on a previous study, purified Δ12 FADs was discovered at the expected size of 49 kDa, with a concentration of only 0.728 mg/mL of culture (Halim et al. 2021). However, a study reported that the concentration of 4.6 mg/L of culture for Δ15 FADs 22.5 mg/L of culture for Δ12 FADs and 37.5 mg/L of culture Δ9 FADs could be obtained through protein solubilization and affinity purifications (Chen et al. 2013). Therefore, there is a need to optimize a few parameters to acquire the desired concentration of crystallization because some membrane proteins may produce crystals at 1 to 2 mg/mL, while others may need a concentration of 20 to 30 mg/mL to crystallize (Li et al. 2014).

Table 2 The comparison of solved structure and predicted structure of Δ9 desaturase in different organisms. Sources from Wang et al. (2015a, b), Bai et al. (2015), Garba et al. (2018), and Shen et al. (2020)
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Joseph E. Stukey (1990), suggested a topological model for membrane-bound FADs which had four trans-membrane spanning domains with N and C termini in the cytosolic side of the membrane, as well as a catalytic site based on Δ9 stearoyl-CoA FADs in yeast and rat. The amino acid sequences of Δ9 bacterial acyl-lipid FADs contained conserved histidine boxes predicted as “HXXXH,” “HXXHH,” and “HXXHH,” which were identical to those found in mammal, yeast, and fungal cells (Fukuchi-Mizutani et al. 1995, 1998). Alignments of Δ9 acyl-CoA FADs in Pseudomonas sp. AMS8 (Garba et al. 2018) and sequences from several other Δ9 FADs (Alberts et al. 2002) revealed three conserved-histidine boxes at positions 34–39, 71–75, and 206–210, which are common to all membrane-bound FADs in bacteria (Alberts et al. 2002; Pereira et al. 2004), fungi (Wang et al. 2015a, b), and animals (Bai et al. 2015). Membrane-bound FADs are thought to have different active sites than soluble desaturases, with a cleft into which substrates enter laterally rather than deep binding cavity; thus, substrates enter in extended conformation as soluble FADs do (Shanklin et al. 2009). The putative active site for Δ9 acyl-CoA FADs in Pseudomonas sp. AMS8 was discovered within the conserved histidine-box region, which had previously been identified as a probable binding site for this enzyme (Garba et al. 2018). Certain residues, consisting of Ile, Tyr, Val, Gly, Pro, Glu, His, Arg, Thr, and Lys were found to bind the palmitic acid, which acts as a substrate for this Δ9 acyl-CoA FADs (Garba et al. 2018). In the crystal structures of Δ9 mSCD1and Δ9 hSCD1, amino acid residues, Ile, Val, Gly, and Arg, are comparable to binding residues (Wang et al. 2015a, b).

A study revealed that three transmembrane domains traverse the membrane bi-layer twice, with both protein termini towards the cytoplasm, using CCTOP to predict the structure of Pseudomonas sp. AMS8 Δ9 acyl-CoA FADs, whereby each domain of TM1, TM2, and TM3 has 20 (Leu13-Leu33), 25 (Leu135-Ile159), and 20 (Met162-Tyr181) amino acid residues, respectively (Garba et al. 2018). This is contrary to the predicted structures of Δ9 mSCD1 (Bai et al. 2015) and Δ9 hSCD1 that had a stem of four transmembrane proteins in each structure (Wang et al. 2015a, b). The topology of Δ9 hSCD1 crystal structure matches the prior predictions of the topology of Δ9 mSCD1. For Δ9 mSCD1, the trans-membrane α-helices (TM) are built in a cone-like shape, where TM1 and TM2 sandwich TM4. TM2 and TM4 protrude, and the membrane makes three helical twists on the cytoplasmic side, which offers a few amino acids for the di-metal active site (Bai et al. 2015). Δ9 hSCD1 exposes four α-helices of trans-membrane and a cytoplasmic domain, which has mushroom-like architecture (Wang et al. 2015a, b). TM1–TM4 of the TM stem apparently span the ER membrane (Fig. 1). The TM stem joins through two short ER-lumen loops (LL1 and LL2) and a long polypeptide linkage, which extends into the cytoplasmic side of the ER (Wang et al. 2015a, b).

Fig. 1
figure 1

Overview of transmembrane helices of hSCD1. Structure of the transmembrane α-helices (TM) was arranged in a cone-like shape. It folds like a mushroom architecture consisting four TM, numbered TM1, TM2, TM3, and TM4

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For acyl-lipid FADs, according to the projected positioning of Δ12 acyl-lipid FADs in relation to the membrane, the enzyme traverses the membrane four times and exposes three histidine clusters that comprise the desaturase catalytic center (Los and Murata 1998). A study suggested that the structure of acyl-lipid FADs is like acyl-CoA FADs, as they are mainly built up of transmembrane proteins (Murata and Wada 1995). Synechocystis sp. PCC 6803’s Δ12 acyl-lipid FADs revealed that a conserved histidine residue substituted by different amino acid residues might lead to the loss of active enzyme as the residues are unable to bind to ferric iron where it is needed (Avelange-Macherel et al. 1995; Schneider et al. 1992). The residues Lys, Glu, and His are also present in probable acyl-ACP FAD genes desA2, with the metal-binding ligands of a plant Δ9 FADs (Alberts et al. 2002). Δ12 acyl-lipid FADs from Synechocystis sp. PCC 6803 had four transmembrane domains, exposing three histidine-box motifs to the cytoplasmic side (Avelange-Macherel et al. 1995) like the crystal structure. However, Δ5 acyl-lipid FADs from Bacillus subtilis was reported with six transmembrane domains (two additional from the proposed topology of membrane desaturase in bacteria) and one membrane-associated domain, which likely represent a substrate-binding motif with three conserved histidine motifs found on the membrane of cytoplasm (Diaz et al. 2002). The presence of two additional trans-membrane domains might be essential for the desaturation of acyl chain in the activity of acyl-lipid desaturase (Yeagle 2016).

Catalytic mechanisms

The catalytic sites of membrane-bound FADs are often situated on the cytosolic side of the membrane, where they have access to substrates and co-factors such as NADH, NADPH, AMP, and ATP (Wang et al. 2013). The predicted structure of Δ9 FADs in Pseudomonas sp. AMS8 revealed that three conserved-histidine boxes found in all classes of these enzymes play a role in binding to two irons at the catalytic core (Garba et al. 2018). Site-directed mutagenesis of rat stearoyl-CoA Δ9 FADs established the importance of these histidine residues in the conserved histidine-box motifs, while residues bordering the conserved area exhibit essential catalytic features in plant FAD2 desaturases and similar enzymes (Meesapyodsuk et al. 2007). Two metal-binding sites in the cytoplasmic cap domain of Δ9 hSCD1 have been discovered. Zn1+ coordinated with five histidine residues: His120, His125, His157, His161, and His301, whereas Zn2+ coordinated with four histidine residues: His160, His269, His298, and His302, as well as a water molecule (Wang et al. 2015a, b) as shown in Fig. 2. The two zinc ions were within 4.5–5.5 Å of stearoyl-CoA carbons 9 and 10, identifying the Δ9 hSCD1 dehydrogenation catalytic center. Two zinc metal ions have also been found in Δ9 mSCD1, with the first zinc surrounded by four histidine residues and the second zinc surrounded by five histidine residues (Wang et al. 2015a, b). The positions of the first and second zinc are identified as 5.2 Å from carbon 9 and 4.7 Å from carbon 10, respectively, while the positions of nine histidine residues are highly conserved (Bai et al. 2015). According to a study, eight histidine residues correspond to three conserved histidine residue motifs, which are one of the characteristics of membrane-bound desaturase (shown in Table 1), alkane hydroxylase, and xylene monooxygenases (Sperling et al. 2003; Shanklin and Cahoon 1998). In addition, with one missing ligand, the coordination of both zinc ions was consistent with an octahedral shape. Nagao et al. (2019) stated that nine conserved histidine residues in humans (His120, His125, His157, His160, His161, His298, His301, and His 302), in mouse (His116, His121, His153, His156, His157, His265, His297, His298), and one conserved Asparagine residue (Asn265 in human; Asn261 in mouse), respectively, in TM2, TM4, the cytosolic loop between TM2 and TM3, and the C-terminal domain. However, substitution of a single histidine residue among the conserved histidine residues eliminates the enzyme’s ability to complement the growth defects (Shanklin et al. 1994).

Fig. 2
figure 2

The di-metal center of zinc-bound hSCD1 coordinated by 9 histidine conserved motifs; Zn1 is coordinated with five histidine residues, while Zn2 is coordinated with four

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The sequence identity of Δ9 hSCD1 and Δ9 mSCD1 is 84% similar, and the structures of both are almost identical to each other with an RMSD of 0.35 Å (Shen et al. 2020). However, the structural fold of SCD1 is different compared to soluble acyl-ACP desaturase that catalyzes a comparable reaction (Behrouzian et al. 2002; Whittle et al. 2008). A study suggested that the existence of two zinc ions, instead of two iron ions, in the catalytic center might cause the enzymes to be inactive. According to a study published in Δ9 mSCD1, the distance between the two metal ions is 6.4 Å, which is the largest distance between two metal ions ever observed in any soluble di-iron enzymes’ solved structures (Hogbom et al. 2002; Lindqvist et al. 1996; Sazinsky and Lippard 2006). Therefore, a recent work was undertaken to insert iron into Δ9 mSCD1 and the structure was resolved by molecular replacement, by utilizing the preceding Δ9 mSCD1; the search model with the presence of both metal ions and histidine residues was at 3.5 Å (6WF2) (Shen et al. 2020). A study revealed that the structures of Δ9 mSCD1 and Δ9 hSCD1 were almost similar, whereby the structure of mSCD1 with insertion of iron was nearly identical to Δ9 mSCD1’s prior structure, with RMSD of 0.3 Å for all backbone atoms (Shen et al. 2020). The solved structure also exhibited the di-metal center, coordinated by the conserved histidine residues, which also portrayed similar coordination geometry (Bai et al. 2015). Furthermore, as Zn2+ has the ionic radius and charge property like Fe2+, it can be used as a suitable surrogate to maintain structural integrity; misincorporation of Zn2+ fails to accelerate the enzyme reaction mechanism. However, it is not unique to detect Zn2+ in the sites that bind iron; few reports suggested the presence of Fe2+ in the structure of yeast fatty acid α-hydroxylase (Zhu et al. 2015) and quinol-dependent nitric oxide reductase (PDB 3AYF) (Hino et al. 2010). A study revealed that Zn2+ has an ionic radius of 0.88 Å, whereby the size and charge are similar to Fe2+ (0.92 Å), so it might have the potential to be a substitute (Shen et al. 2020). Through comparisons of the di-iron clusters of several di-iron-containing enzymes, relevant information about the tuning of di-iron centers about various chemical reactivities has been made available (Shanklin and Cahoon 1998; Shanklin et al. 2009; Soon and Lippard 2004). This indicates that the iron-bound oxidant is present in the experiment (Shanklin and Cahoon, 1998).

Stearoyl-CoA binding site

Desaturation begins with the energy-intensive abstraction of hydrogen from a methylene group using stearoyl-CoA as the substrate, which results in the insertion of double bonds within the fatty acyl chain (Shanklin et al., 2009) (shown in Fig. 3). Multiple proteins share an active-site di-iron cluster in this process, including methane monooxygenase, ribonucleotide reductase, rubrerythrins, and a variety of oxidase enzymes to recruit and activate molecular oxygen (Shanklin et al. 2009; Soon and Lippard 2004). A study reported the interaction between SCD1 with the stearoyl-CoA, in which the polar residues of the surface of SCD1 guide the fatty acyl chain towards the tunnel located between TM2 and TM4 (Wang et al. 2015a, b). The substrate must induce a cis conformation between C9 and C10 before it aligns proximally at the di-metal center for the fatty acyl chain to fit the tunnel (Wang et al. 2015a, b). The di-metal center’s relative orientation to C9 and C10 is necessary for the reaction of pro-R dehydrogenation, which demands additional elements (Enoch et al. 1976). The CoA groups (adenosine, diphosphate, and pantothenate) interact with the polar remains on SCD1’s surface via hydrogen-bonding and electrostatic interactions in this CoA-SCD1 interface (shown in Fig. 4). The adenosine group of CoA mainly forms hydrogen-bonding connections with the side chains of residues Arg155 and Asp156 from CH2 and with the main chain carbonyl group of Gly197 (Wang et al. 2015a, b).

Fig. 3
figure 3

The binding site with the presence of steaoryl-CoA. Five histidine and four histidine residues coordinate the presence of two zincs, Zn1 and Zn2, showed the interaction with the substrate

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Fig. 4
figure 4

Electrostatic surface of the hSCD1 structure, which portrayed contact of steaoryl-CoA with hSCD1. It is showing the positive potential in the steaoryl-CoA head group-binding site

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Current study and future perspectives of FADs

Cellular biology and biochemistry make membrane protein an interesting subject of study. It represents between 20 and 30% of the proteins of most organisms and more than 60% of drug targets (Attwood and Schioth, 2021) important for pharmaceuticals, and yet very few structures of these proteins have been solved. Over the last 15 years, membrane protein structural biology has made great leaps despite their instability (Birch et al. 2020). To date, the database of the “Membrane Proteins of Known 3D Structure” has shown an additional 1472 unique membrane protein structures, with 6252 coordinate files of membrane proteins deposited in the Protein Data Bank (PDB) in August 2022. This contrasts with only 2588 coordinated files of membrane proteins deposited in the PDB with a list of 1141 unique membrane protein structures in September 2020 (Birch et al. 2020). As of today, none of the crystal structures of membrane-bound FADs from bacteria has been established and solved. It remains a major problem in determining crystal structures of FADs for some reasons, such as difficulties in expressing and purifying them (Birch et al. 2020), as well as the identifications of suitable conditions for production of crystals for structure determination. However, different strategies, such as using mutant host strains E. coli C41(DE3) and C43(DE3) known as “Walker strain” and shifting towards eukaryotic targets and expression systems might be promising to improve the protein production yield of FADs (Karyolaimos and de Gier 2021). An effective recombinant membrane protein production plan must consider a broad range of factors, including construct design, expression system, extraction, and purification methods (Birch et al. 2020). As more information becomes available, the easier it may become to develop predictors and other computer-aided tools that can assist in designing strategies to enhance protein production yield (Cetnar and Salis 2021). Furthermore, having purified FADs paves the way towards the characterization biochemically and biophysically (Halim et al. 2021) and may be beneficial for the specification of function, structure, and interactions of membrane proteins.

The success of the purification and crystallization of membrane proteins has greatly depended on the detergents’ ability to maintain a target membrane protein in a functional, folded state in the absence of membrane (Kermani 2020). It is crucial to identify the ideal combination of constructs and detergents as it is important to work with stable, correctly folded protein (Birch et al. 2020). The primary method for membrane protein crystallization has relied on detergents' ability and successful crystallization screening kits (Healey et al. 2021) such as MemGold, MemAdvantage, and MemMeso, which are credited to advances in tools and reagents used to manipulate membrane proteins. This improvement has been accompanied by the development of cryogenic electron microscopy (cryo-EM) and X-ray crystallography as 80% of membrane protein structures have been solved by this leading technique (Kermani 2020). Every new discovery in membrane protein structural biology shifts the bottlenecks in a new direction. The understanding of the correlative techniques combining super resolution light microscopy with cryo-correlative light and electron microscopy (cryo-CLEM), cryo-ET, or cryo-X-ray tomography will promote understanding of the structural information of membrane proteins (Birch et al. 2020). These emerging innovations are promising for future research efforts to overcome the intrinsic difficulties and should have a major impact on understanding of membrane proteins as the ability of this electron microscope with advances in cryo-technology may provide high-resolution images of detailed structural and chemical information about proteins.

Conclusion

In summary, this manuscript shows that FADs have been isolated from all groups of organisms. Class of soluble desaturases has been extensively studied, whereas the membrane-bound class of desaturases lacks sufficient information on its structure–function relationships due to its difficult nature to purify. Biosynthesis of FADs revealed that this enzyme is responsible for the production of polyunsaturated fatty acids through the mechanism of desaturation reaction. Fatty acid desaturation involves an enzymatic reaction in which a double bond is introduced into an acyl chain, and a molecule of oxygen is completely reduced to water. Role of unsaturated FADs tend to exert a variety effect in microorganisms, plants, humans, and other mammals. The 3D structures of FADs demonstrate almost similar structural architecture, specifically in humans and mammals, with four transmembrane helices arranged in a cone shape. FADs possess a unique di-iron catalytic site, which occurs due to different di-iron metal ions; however, they can maintain the integrity of the structure.