Structure and Function of Δ9-Fatty Acid Desaturase

Vol. 67, No. 4 Chem. Pharm. Bull. 67, 327–332 (2019) 327 Current Topics

Recent Progress in Biophysical Research of Biological Membrane Systems

Review

Kohjiro Nagao,* Akira Murakami, and Masato Umeda Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University; Kyoto 615–8510, Japan. Received December 19, 2018

Δ9-Fatty acid desaturase (Δ9-desaturase) is a rate-limiting enzyme of unsaturated fatty acid biosynthesis in animal cells and specifically introduces a cis-double bond at the Δ9-position of acyl-CoA. Since the chemical structure of fatty acids determines the physicochemical properties of cellular membrane and modu- lates a broad range of cellular functions, double bond introduction into a fatty acid by Δ9-desaturase should be specifically carried out. Reported crystal structures of stearoyl-CoA desaturase (SCD)1, one of the most studied Δ9-desaturases, have revealed the mechanism underlying the determination of substrate preference, as well as the position (Δ9) and conformation (cis) of double bond introduction. The crystal structures of SCD1 have also provided insights into the function of other Δ9-desaturases, including Drosophila homologs. Moreover, the amino-terminal sequences of Δ9-desaturases are shown to have unique roles in protein degradation. In this review, we introduce recent advances in the understanding of the function and regulation of Δ9-desaturase from the standpoint of protein structure. Key words unsaturated fatty acid; phospholipid; crystal structure; substrate specificity; protein degradation

1. Introduction lular lipids. Fatty acid desaturases are a family of enzymes that intro- Recently reported crystal structures of mammalian duce cis-double bonds into the acyl chain of acyl-CoA. There stearoyl-CoA desaturase (SCD)1, one of the most studied are several types of fatty acid desaturases having a variety of Δ9-desaturases, demonstrated the mechanism underlying the substrate preferences and specificities for the position of dou- determination of substrate preference, as well as the position ble bond introduction. Among them, Δ9-fatty acid desaturase (Δ9) and conformation (cis) of double bond introduction. Fur- (hereafter referred to as Δ9-desaturase), which is embedded in thermore, the amino acid sequences of Δ9-desaturase that are the membrane of the endoplasmic reticulum (ER), introduces responsible for the regulation of protein degradation have been a cis-double bond exclusively at the Δ9 position of acyl-CoA1) identified. In this review, we introduce recent advances in the (Fig. 1). This reaction requires molecular oxygen and electrons understanding of the function and regulation of Δ9-desaturase derived from electron relay systems via cytochrome b5, cyto- from the standpoint of protein structure. chrome b5 reductase, and nicotinamide adenine dinucleotide (phosphate) (NAD(P) H)2) (Fig. 1). 2. Position and Conformation of Double Bond Intro- Δ9-Desaturase is a rate-limiting enzyme involved in the duced by Δ9-Desaturase biosynthesis of monounsaturated fatty acids that are used Biochemical studies have predicted that the amino- (N) to synthesize polyunsaturated fatty acids, phospholipids, and carboxy- (C) termini of SCD1 are oriented toward the triacylglycerols, cholesteryl esters, and wax esters. The fatty cytosol, with four transmembrane helices (TMs) separated by acid double bond affects the properties and functions of fatty two short hydrophilic loops in the ER lumen and one large acid-containing lipids.3) For example, the number and position hydrophilic loop in the cytosol.6) Consistent with these bio- of the double bonds in the fatty acid moieties of phospho- chemical results, the reported crystal structures of human and lipids determine the physicochemical parameters of cellular mouse SCD1 have four TMs arranged in a cone-like shape7,8) membranes: unsaturated fatty acid-containing phospholipids (Fig. 2A). The crystal structure has a narrow tunnel extending have lower phase transition temperatures and tend to form approximately 24 Å in which the acyl chain of acyl-CoA is en- membranes with a liquid-disordered phase.4) Recently, Budin closed. The substrate-binding tunnel has a hydrophobic inte- et al. demonstrated that the regulation of unsaturated fatty rior with a sharp kink around the 9th and 10th carbons of the acid biosynthesis in Escherichia coli and budding yeast modu- acyl chain of the bound acyl-CoA. Furthermore, the carbonyl lates membrane viscosity, as well as the activity of electron group of the acyl chain and CoA moiety can be specifically transport chains that feature diffusion-coupled reactions be- recognized via hydrogen bonds and electrostatic interactions. tween enzymes and electron carriers.5) Therefore, the reaction There is a large positively charged surface on the CoA moi- of double bond introduction should be specifically carried out ety-binding site, which is well suited to the recognition of a to maintain the appropriate properties and functions of cel- CoA moiety containing negatively charged phosphate groups.

Fig. 1. Δ9-Desaturase Reaction Δ9-Desaturase introduces a cis-double bond at the Δ9 position of acyl-CoA. The reaction requires molecular oxygen and electrons derived from electron relay systems via cytochrome b5, cytochrome b5 reductase, and NAD(P)H.

Fig. 2. Structure of SCD1 Protein (A) Structure of mouse SCD1 (PDB ID: 4YMK).7) Two metal ions are shown as black spheres. The structure of bound acyl-CoA is also shown. (B) Residues of mouse SCD1 involved in the recognition of acyl-chain and the coordination of metal ions.7) Two metal ions are shown as black spheres. The structure of bound acyl-CoA and the side chain of Tyr104 and Ala108 are shown. The side chains of conserved histidine and asparagine residues in the dimetal center are also shown.

This speciﬁc recognition of acyl-CoA enables the enzyme to has been reported that substitution of a single histidine residue determine the arrangement of acyl chain of bound acyl-CoA. among conserved eight histidine residues in rat SCD1 (cor- It is easy to assume that this narrow tunnel with its sharp kink responding to His116, His121, His153, His156, His157, His294, is well suited to the crucial determination of the positon (Δ9) His297, and His298 in mouse SCD1) eliminates the enzyme’s and conformation (cis) of the double bond introduced by Δ9- ability to complement the growth defects of a Δ9-desaturase- desaturase. deﬁcient yeast strain.9) These histidine and asparagine residues There are nine conserved histidine residues (His120, compose the dimetal center, which is adjacent to the kink of His125, H157, His160, His161, His269, His298, His301, and the substrate-binding tunnel (Fig. 2B). In the dimetal center, His302 in human SCD1; His116, His121, His153, His156, two metals are coordinated by the nine nitrogen atoms on the His157, His265, His294, His297, and His298 in mouse SCD1) side chains of the histidine residues and one water molecule and one conserved asparagine residue (Asn265 in human that interacts with carbonyl group on the side chain of the SCD1; Asn261 in mouse SCD1) in TM2, TM4, the cytosolic asparagine residue (Fig. 2B). Although two zinc ions are de- loop between TM2 and TM3, and the C-terminal domain. It tected in the reported crystal structures of human and mouse Vol. 67, No. 4 (2019) Chem. Pharm. Bull. 329

SCD1, this is expected to be an artifact of protein overexpres- SCD3 to a stearoyl-CoA-preferring Δ9-desaturase.7) Because 7,8) sion. There are several reasons why the coordinated ions in the side chain of isoleucine (–CH(CH3)–CH2–CH3) is larger native SCD1 protein are expected to be iron ions rather than than that of alanine (-CH3), it is likely that the large hydro- zinc ions: i) although zinc ion normally has a tetrahedral coor- phobic side chain of isoleucine hinders the binding of stearoyl- dination, coordinated ions in SCD1 structures have octahedral CoA, but not that of palmitoyl-CoA in mouse SCD3. coordination, which is the typical form for the coordination of These substrate-determining residues at the end of the iron ion; ii) Fe2+ (0.92 Å) and Zn2+ (0.88 Å) have similar ionic substrate-binding tunnel may also play a role in the unique radii; and iii) the diiron center is widely observed in a variety substrate preference of Drosophila Δ9-desaturaes. The Dro- of oxidase enzymes including soluble Δ9-stearoyl-acyl carrier sophila genome contains the Δ9-desaturase-encoding genes protein (ACP) desaturase.10–12) Desat1 and Desat2. DESAT1 and DESAT2 comprise 383 and To introduce a cis-double bond into the acyl chain of acyl- 361 amino acids with four TMs, and show structural features

CoA, Δ9-desaturase accepts electrons from cytochrome b5 similar to those of SCD1. DESAT1 was identified by its ho- 14) (Fig. 1). It is likely that cytochrome b5 binds to SCD1 in the mology to vertebrate fatty acid desaturases and subsequent vicinity of the dimetal center to effectively transfer electrons genetic studies have revealed the role of DESAT1 in the because the dimetal center is accessible from the cytoplas- control of sensory communications via pheromone produc- mic side. It is estimated that the positively charged surface tion, as well as regulation of the double bond contents in the 15–17) of SCD1 and negatively charged surface of cytochrome b5 acyl chains of phospholipids. DESAT2 was identified complement each other.7) To reveal the exact mechanism of through genomic screening for the enzyme which determines electron transfer and double bond formation, an understanding the population-specific composition of female cuticular phero- 17) of the complex structure of Δ9-desaturase and cytochrome b5 mone. There is a considerable variety in the composition of is required. Drosophila melanogaster female cuticular pheromones, which are composed of cis-double bond-containing hydrocarbons 3. Substrate Preference in Δ9-Desaturase and are produced from unsaturated fatty acids. Female cuticu- The mechanism underlying the determination of acyl- lar pheromones from African and Caribbean populations have chain length of the substrate acyl-CoA is also revealed by high ratios of 5,9-heptacosadiene/7,11-heptacosadiene, whereas SCD1 crystal structures. It has been reported that SCD1 this ratio is low in populations from other areas. Genomic prefers acyl-CoA with lengths of 17-, 18-, and 19-carbons.2) analysis revealed that DESAT2 is not expressed in popula- Mouse SCD1 has Tyr104 and Ala108 residues at the end of tions other than those of Africa and the Caribbean because the substrate-binding tunnel (Fig. 2B). Notably, the hydroxyl of a 16-bp deletion in the 5′ region of the Desat2 gene.18) group of Tyr104 is located close (4.1 Å) to the methyl group of When DESAT1 is expressed in a Δ9-desaturase-deficient yeast the acyl chain of stearoyl-CoA in mouse SCD1. This tyrosine strain, palmitoleic acid (C16:1) and oleic acid (C18:1) are the residue is widely conserved in human, mouse, and Drosophila monounsaturated fatty acids that are primarily produced.17) Δ9-desaturases (Fig. 3). Mouse SCD3 is also called palmitoyl- On the other hand, DESAT2-expressing yeast produces mainly CoA-desaturase, and prefers palmitoyl-CoA (C16-carbon myristoleic acid (C14:1), with only trace amounts of C16:1 length acyl-CoA) rather than stearoyl-CoA (C18-carbon and C18:1.17) Because C14:1 and C16:1 are required for the length acyl-CoA) as a substrate.13) Mouse SCD3 has Tyr108 production of 5,9-heptacosadiene and 7,11-heptacosadiene, and Ile112 at the sites corresponding to Tyr104 and Ala108 respectively, functional expression of DESAT2 is required of mouse SCD1, respectively (Fig. 3). Substitution of alanine for the production of female cuticular pheromone with a high for isoleucine at position 112 together with three (Glu113Leu, ratio of 5,9-heptacosadiene/7,11-heptacosadiene. Interestingly, Asp281Gln, and Pro282Ser) or four (Glu113Leu, Val119Ala, DESAT2 has a Met91 residue at the site corresponding to Asp281Gln, and Pro282Ser) other mutations convert the mouse that of the Ala108 residue in mouse SCD1 (Fig. 3). Reflecting on the similarity of the substrate preference between SCD1 and DESAT1, DESAT1 has a Gly112 residue at the site corresponding to that of the Ala108 residue of mouse SCD1 (Fig.

3). Because the side chain of methionine (–CH2–CH2–S–CH3; DESAT2) is larger than that of alanine (–CH3; mouse SCD1), glycine (–H; DESAT1), or isoleucine (–CH(CH3)–CH2–CH3; mouse SCD3), differences in the substrate preference of Dro- sophila Δ9-desaturases may be explained by the side chain of the amino acid residue at the end of the substrate-binding tunnel. In fact, a DESAT2-Met91Gly mutant effectively produces C16:1 and C18:1 (Miyamoto K., Nagao K., Umeda M. unpublished observation). Unsaturated fatty acids with different chain lengths are reported to have different roles not only in Drosophila, but also in mammalian models (i.e., C16:1 Fig. 3. Residues Involved in the Determination of Δ9-Desaturase Sub- is called lipokine, a lipid hormone linking adipose tissue to strate Speciﬁcities systemic metabolism).19,20) Comparison of the substrate prefer- Residues comprising the end of the substrate-binding tunnel in Δ9-desaturases (highlighted in black) from various species were compared: Homo sapiens ence of a wide range of Δ9-desatuarses based on the amino SCD1 (NP_005054.3), H. sapiens SCD5 (NP_001032671.2), Mus musculus acid residues at the end of the substrate-binding tunnel will SCD1 (NP_033153.2), M. musculus SCD2 (NP_033154.2), M. musculus SCD3 (NP_077770.1), M. musculus SCD4 (NP_899039.2), Drosophila melanogaster facilitate our understanding of the physiological roles and the DESAT1 (NP_652731.1), and D. melanogaster DESAT2 (NP_650201.1). synthesis pathway of each unsaturated fatty acid molecule 330 Chem. Pharm. Bull. Vol. 67, No. 4 (2019)

Fig. 4. N-Terminal Sequences of Δ9-Desaturases The N-terminal amino acid sequences of Δ9-desaturases from various species were compared: H. sapiens SCD1 (NP_005054.3), M. musculus SCD1 (NP_033153.2), D. melanogaster DESAT1 (NP_652731.1), and D. melanogaster DESAT2 (NP_650201.1). Proline, glutamic acid, serine, and threonine are highlighted in gray. The di-proline motif of DESAT1 is enclosed. with a different chemical structure. the 33 N-terminal residues of SCD1 and GFP has a short half- life when expressed in the cytosol, but not in the ER lumen, 4. Regulation of the Δ9-Desaturase Expression Level demonstrating that the N-terminal cytosolic domain of SCD1 Expression levels of mammalian SCD1 are regulated at has a rapid degradation signal for the cytosol proteases.29) the transcriptional level by sterol regulatory element-binding Using a biochemical approach, Heinemann et al. identified protein (SREBP), carbohydrate-responsive element-binding a plasminogen-like protein as a protease for the microsomal protein, liver X receptor, and insulin signaling.21) Similar degradation of SCD1 protein.30,31) They also demonstrated that to SCD1, expression levels of Drosophila DESAT1 are also expression level of SCD1 in liver microsomes is decreased in regulated at the transcriptional level.22) There are five splicing plasminogen-deficient mice.30) variants that are transcribed from different upstream regions The N-terminal sequence of SCD1 contains a tag for rapid of the Desat1 gene, but encode the same amino acid sequence. protein degradation, which is called PEST sequence and en- Tissue-specific expression of the Desat1 gene is precisely reg- riched in proline, glutamic acid, serine, and threonine32,33) ulated by distinct putative regulatory regions targeting either (Fig. 4). Kato et al. reported that degradation of endogenously pheromone biosynthetic cells, neurons involved in pheromone and exogenously expressed SCD1 protein is suppressed by perception, or non-neuronal cells.23) It has also been reported proteasome inhibitors (MG132 and epoxomicin).33) Further- that the expression of Desat1 gene in pheromone biosynthetic more, in MG132-treated cells, SCD1 protein is poly-ubiqui- cells called oenocytes is under the control of circadian clock tinated and interacts with AAA-ATPase p97, indicating that genes, which affects pheromone production and mating behav- SCD1 is degraded via the ER-associated degradation path- ior in Drosophila.24) way.33) Moreover, they demonstrated that the 66 N-terminal Although the expression levels of Δ9-desaturases could residues containing the PEST sequence are important for the be intensively regulated at the transcriptional level, Δ9- proteasomal degradation of ER-localizing proteins.33) desaturases have intrinsic motifs regulating their degradation The expression level of Δ9-desaturase should be strictly in their N-terminal domains. In contrast to the highly con- regulated to maintain the appropriate physicochemical proper- served TMs, the cytosolic loop, and the C-terminal domain, ties of cellular membranes. Although the regulatory mecha- the N-terminal cytosolic domain has low homology between nisms of the expression of several fatty acid desaturases are human, mouse, and Drosophila Δ9-desaturses (Fig. 4). Sec- reported, it is unclear how changes in the level of cellular ondary structure prediction program25) suggests that the N- unsaturated fatty acids are recognized by intracellular ma- terminal domains of DESAT1 and mouse SCD1 do not have chinery to regulate the expression of fatty acid desaturase. apparent secondary structures. Furthermore, the structure While the degradation rate of SCD1 protein is irrespective of the N-terminal domain of human and mouse SCD1 is not of the cellular levels of unsaturated fatty acids,33) we recently completely solved in reported crystal structures.7,8) Therefore, reported that the expression level of Δ9-desaturase is post- it is thought that the N-terminal domains of Δ9-desaturases translationally regulated by the cellular fatty acid composition have a variety of regulatory motifs for post-translational regu- in Drosophila melanogaster.34) Drosophila melanogaster is lation such as protein degradation. a model organism that provides advantages for the study of Since the degradation rate of SCD1 in the microsome frac- mechanisms underlying the expression and function of Δ9- tion is fast, with a half-life of only a few hours, SCD1 is re- desaturase.34) First, DESAT1 is the sole fatty acid desaturase garded as a short-lived protein.26–28) Because the transcription that introduces a cis double bond into acyl chain of acyl-CoA of SCD1 could be strictly regulated, the short life of protein in typical Drosophila cell lines such as S2 cells because an- is well suited to the finely tuned regulation of SCD1 expres- other fatty acid desaturase, DESAT2, is not expressed.18) Sec- sion. It has been reported that C-terminally green fluorescent ond, because Drosophila cannot synthesize sterols, and only protein (GFP)-tagged rat SCD1 protein expressed in CHO-K1 a trace amount of polyunsaturated fatty acids are detected in cells has a half-life of a few hours.29) However, N-terminal cellular phospholipids,34–36) changes in Δ9-desaturase activity truncated SCD1 proteins consisting of residues 27–358 or are expected to directly affect the physicochemical properties 45–358 of SCD1 are stable.29) Furthermore, a fusion protein of of the cellular membrane. Therefore, Drosophila provides a Vol. 67, No. 4 (2019) Chem. Pharm. Bull. 331 useful model to study how cells recognize changes in the level against high-fat diet-induced obesity may be explained by its of cellular unsaturated fatty acids and regulate the expression effect on the skin. In contrast, high-carbohydrate diet-induced of Δ9-desaturase. adiposity is prevented in liver-specific SCD1-deficient mice.43) DESAT1 protein is rapidly degraded in Drosophila S2 cells, Moreover, hepatic lipogenesis and levels of active SREBP1 are with a half-life of approximately 2 h,34) which is comparable to decreased in liver-specific SCD1-deficent mice on a high-su- those observed for mammalian SCD1 proteins. The degrada- crose very low-fat diet,43) indicating that SCD1 plays a pivotal tion of DESAT1 protein is significantly enhanced by supple- role in hepatic lipogenesis. mentation of culture medium with unsaturated fatty acids Inhibition of Δ9-desaturase is an attractive target for but not saturated fatty acids, in which exogenously added therapeutic intervention in cancer because the production of fatty acids are rapidly incorporated into cellular phospho- monounsaturated fatty acids is required for the replication and lipids.34) Furthermore, Δ9-desaturase inhibitors37,38) decrease survival of mammalian cells.39) In proliferating cells, the ex- the amount of unsaturated fatty acyl chain of phospholipids, pression of SCD1 is upregulated by the activation of SREBP1, and significantly suppress the degradation of DESAT1,34) sug- a target of the phosphatidylinositol 3-kinase (PI3K), Akt, and gesting that changes in the composition of the acyl chains of mechanistic target of rapamycin (mTOR) pathways.45) Fur- phospholipids are responsible for regulation of the expression thermore, inhibition of SCD1 causes the suppression of pro- level of DESAT1. liferation and survival signaling in cancer cells.46,47) However, We found that the two sequential proline residues in the because SCD1 is required for a wide range of physiological N-terminal sequence Met1-Pro2-Pro3-Asn4-Ala5-Gln6 are functions, including lipogenesis in liver and skin, it should responsible for unsaturated fatty acid-dependent degradation be noted that SCD1 inhibitors may have harmful side effects. of DESAT1; single mutations (Pro2Ala and Pro3Ala), as well Recently, tumor-specific irreversible inhibitors of SCD1 were as a double mutation (Pro2,3Ala) in DESAT1 remarkably discovered.48) Sensitive cell lines against these compounds ex- abolish the responsiveness of DESAT1 protein degradation press CYP4F11 and metabolize these compounds into irrevers- to the level of fatty acid desaturation.34) In the light of these ible inhibitors of SCD1.48) Because these compounds are not findings, we designated the sequential prolines (Pro2–Pro3) of activated in sebocytes, which do not express CYP4F11, toxic- DESAT1 as a di-proline motif, which is crucial for the regu- ity in the skin can be avoided.48) Although specific interaction lation of DESAT1 expression in response to changes in the between SCD1 and these inhibitors is reported, the binding level of cellular unsaturated fatty acids.34) By comparing the site and mechanism for SCD1 inhibition have not been eluci- N-terminal sequences of Δ9-desaturases, we found that most dated. Structure-based evaluation and development of SCD1 Δ9-desaturases have a single proline residue at N-terminal po- inhibitors will be facilitated by reported crystal structures of sition 2 or 3, except DESAT1 with its di-proline motif34) (Fig. SCD1, as well as further studies of Δ9-desaturases using bio- 4). Interestingly, DESAT2, which is different from DESAT1 in physical, biochemical, and biological approaches. substrate preference and physiological function, does not have a di-proline motif. Introduction of Pro2–Pro3 residues into the Acknowledgments This work was supported by Grant- N-terminus of mouse SCD1 significantly enhances the unsatu- in-Aid for Scientific research 15H05930 (to M. U.), 15K21744 rated fatty acid-dependent degradation of mouse SCD1 in S2 (to M. U.), 18K19296 (to M. U.), 17H03805 (to M. U.), and cells,34) demonstrating that the di-proline motif works in the 18K05433 (to K. N.) from Japan Society for the Promotion of context of mammalian Δ9-desaturase protein. Furthermore, Science (JSPS) and Ministry of Education, Culture, Sports, genetics and pharmacological analyses demonstrated that cal- Science and Technology (MEXT) of Japan. pain A and calpain B, typical calpains containing motifs for Ca2+ and lipid binding, are involved in the di-proline motif- Conflict of Interest The authors declare no conflict of mediated degradation of Δ9-desaturase protein.34) interest.

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