Vol. 67, No. 4 Chem. Pharm. Bull. 67, 327–332 (2019) 327 Current Topics Recent Progress in Biophysical Research of Biological Membrane Systems Review Structure and Function of Δ9-Fatty Acid Desaturase 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 biosyn- thesis 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 degra- dation. 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. * To whom correspondence should be addressed. e-mail: [email protected] © 2019 The Pharmaceutical Society of Japan 328 Chem. Pharm. Bull. Vol. 67, No. 4 (2019) 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.