Introduction

Stearoyl-CoA Desaturase is an enzyme essential for the biosynthesis of monosaturated fatty acids from saturated fatty acids ^[1]. SCD catalyzes the rate-limiting step in the conversion of Stearoyl-CoA to oleic acid (Fig 1), an essential substrate in the biosynthesis of phospholipids, triacyclglycerols, and cholesterol ^[2]. SCD is highly conserved in eukaryotes and has different gene isoforms. Mice have four isoforms: SCD1, SCD2, SCD3, and SCD4. Humans have two different isoforms: SCD1 and SCD5. The SCD isoform discussed in this page is Stearoyl-CoA Desaturase 1 (SCD1) found in mice. SCD is believed to have once been an anaerobic pathway found in cartilaginous fish about 450 million years ago ^[3]. The enzyme’s mechanism is now aerobic and this aerobic pathway is favored. The structure of SCD1 was found using X-ray crystallography ^[2].

[Figure 1] Desaturation Reaction Catalyzed by SCD1: Stearoyl-CoA (left) is converted to oleoyl-CoA (right) through the introduction of a double bond between C9 and C10.

Structure

Overall Structure

[Figure 2] SCD1 and Electron Transport Chain: Two electrons from NADH (left) are transported to Cytochrome B5 Reductase (Green) then Cytochrome B5 (Blue) and finally to SCD1 (Right) where they can be used in the desaturation reaction. Cytochrome B5 Reductase and Cytochrome B5 are bound to the cytosolic side of the ER membrane whereas SCD1 is embedded within the membrane of the ER.

Its substrate, Stearoyl-CoA, binds to the cytosolic region which contains a "kink" that properly orients Stearoyl-CoA to undergo a dehydrogenation reaction between the of Stearoyl-CoA.

Using X-ray crystallography, three structures of SCD have been found, differing only in their dimetal center and organism of origin. One structure includes the substrate (Stearoyl CoA) a water molecule, and two ions in the center (4YMK) ^[2]

A second structure includes the product (Oleic Acid) and two ions in the center (6WF2). When testing the Zn centered structure, the enzyme was found to be inactive ^[4] The Zn ions serve as a surrogate for Fe as it has similar characteristics including charge and ionic radius size ^[5] .However, Zn did not display its typical coordination geometry, tetrahedral; instead, it displayed octahedral geometry which is common of Fe ion coordination ^[2] More recently, the structure of the human SCD1 (hSCD1) protein was found. This structure was found with Zn incoordination; however, the researchers agree that iron is the true metal in the dimetal center. ^[6] For the images and links below, the zinc ion centered structure will be used as it includes the substrate, even though iron is confirmed as the dimetal center.

Binding of Substrate

Stearoyl-CoA is the substrate that binds to the enzyme, SCD1. The binding of the substrate is stabilized by specific residues on the exterior and interior of the protein. Stearoyl-CoA is a long-chain fatty acyl-CoA. The head group of the substrate is composed of an adenine, ribose, phosphate groups, and polar atoms such as of nitrogen, oxygen, and sulfur. The head of stearoyl-CoA is attached to the exterior of the protein by polar residues. The adenine, ribose, and phosphate are attached by the residues . The remaining exterior of the substrate is attached by the residues ^[2]. All the conserved residues are attached to the Stearoyl-CoA via hydrogen bonds. The fatty acid tail of Stearoyl-CoA is a 17-carbon chain which reaches into the interior of the protein. The fatty acid chain dives into the interior hydrophobic tunnel which is long, narrow, and approximately 24 Angstroms long ^[2]. The geometry of the tunnel and formation of bound acyl chain are the structural basis for the stereospecificity of the desaturation reaction ^[2].

Kink of Chain

The chain is kinked at where the double bond is generated. The kink is induced through the interactions of four conserved residues. Three out of four of these residues are not bound to the chain, but are hydrogen bonded to each other: . T257 is hydrogen bonded to Q143, and Q143 is hydrogen bonded to W149 ^[2]. These residues are directly below the kink and will be hydrolyzed when the enzymatic product is ready to be released. Specifically, if the hydrogen bond between T257 and Q143 is broken, a large opening would allow for the product to be released into the bilayer ^[2]. The residue that is directly hydrogen bonded to the chain is . This residue is highly conserved and stabilizes the chain so it will be in the correct orientation in the active site. The enzyme will be effective on acyl chains that are between 17 to 19 carbons long. The residue that has a role in determining substrate length is . Y104 is a capping residue that has approximately 4 Angstroms between its' hydroxyl oxygen and the end of the chain ^[2].

Active Site

Using X-ray crystallography, two structures of SCD have been found, differing only in their dimetal center. One structure includes the substrate (Stearoyl CoA) a water molecule, and two ions in the center (4YMK) ^[2] A second structure found more recently includes the product (Oleic Acid) and two ions in the center (6WF2). When testing the Zn centered structure, the enzyme was found to be inactive ^[4] The Zn ions serve as a surrogate for Fe as Zn did not display its typical coordination geometry, tetrahedral; instead, it displayed octahedral geometry which is typically of Fe ion coordination ^[2] For the images and links below, the zinc ion centered structure will be used as it includes the substrate, even though iron is confirmed as the dimetal center.

The dimetal center is essential to the catalytic activity, as previously demonstrated in the mechanism above. The ions are 6.4 angstroms apart ^[2]

The ions sit above the kink created by C9 and C10 of the substrate within the active site. The ions are held into the active site through the ^[7] . The nine coordinating His residues stabilize the ions into the active site forming a non-heme prosthetic group ^[7] . The His box is highly conserved among the isoforms of SCD ^[4] . The closest to C10 of the substrate is 4.7 angstroms away from this carbon ^[2] . This ion is coordinated by five histidine residues. The closest to C9 of the substrate is 5.2 angstroms away from this carbon ^[2] This ion is coordinated with four histidine residues and one water molecule. The is in coordination to the zinc ion closest to it. It occupies the fifth .

Residues around the periphery hydrogen bond to the His box to stabilize it. These residues include and ^[2] . Another residue that stabilizes the active site is . This residue hydrogen bonds to the water molecule ^[2] .

The His box and periphery residues stabilize the dimetal center and make up the of the enzyme.This allows for the proposed above mechanism to be carried out.

Mechanism

[Figure 3] Proposed Mechanism: The SCD1-catalyzed desaturation reaction involves a molecular oxygen, water molecule, two protons and electrons, and two iron ions within the enzyme core which through a series of redox reactions and hydrogen transfers introduce a double bond between the 9th and 10th carbons of Stearoyl-CoA forming oleic acid. The penta- and tetra-coordinated irons within the enzyme core are represented by Fe(A) and Fe(B) respectively. All electron pushing steps are shown with reactive groups color coded.

Although the precise mechanism behind SCD1 catalysis is still unknown, several mechanisms have been proposed. In a recent article by Yu and Chen, they propose a novel mechanism for the SCD1 catalyzed desaturation reaction involving a molecular oxygen, water, two protons, and two electrons (2e-). ^[8]

In the first step of the proposed mechanism, water and a molecular oxygen bind to the penta- and tetra-coordinated irons in the active site of SCD1, Fe(A) and Fe(B) respectively (Fig 3). This binding results in the transfer of an electron from the tetra-coordinated iron to the molecular oxygen forming an iron(III)-dioxygen radical species. This is followed by a proton transfer from the iron(II) bound water to the iron(III) bound di-oxygen radical which results in the formation of iron(II)-hydroxyl radical and iron(II)-peroxyl radical intermediates (Fig 3).

One electron and one proton, originating from the electron transport chain, are then incorporated into the reaction resulting in the protonation of the hydroxyl radical to form an iron(II)-water intermediate. In this step the iron(II)-peroxyl radical is coordinated by the iron(II)-water and the tetra-coordinated iron (Fig 3).This step is quickly followed by the dissociation of the O-O bond of the peroxyl radical where one of the hydrogens from the iron(II)-water is transferred to the radical oxygen on the iron(II)-peroxyl resulting in the formation of a triple-hydroxyl intermediate (Fig 3) with the penta-coordinated iron being converted to iron(III).This intermediate then undergoes a hydrogen transfer where a hydrogen from the one of the hydroxyl groups on the dihydroxyl intermediate is transferred to the other hydroxyl group on the intermediate. This results in the formation of a water molecule coordinated between a newly formed high-valent iron(IV)=O and the iron(III)-hydroxyl (Fig 3).

Following the formation of the high-valent iron(IV)=O, the first hydrogen abstraction from the substrate occurs with C9 hydrogen on the substrate being abstracted by the iron(IV)=O forming a C9 radical on the substrate and converting the high-valent iron(IV)=O to a iron(III)-hydroxyl (Fig 3). This is quickly followed by another hydrogen abstraction from the penta-coordinated iron(III)-hydroxyl which results in the formation of a double bond between C9 and C10 and converting the penta-coordinated iron(III)-hydroxyl to iron(II)-water. Once the product has been formed, another proton and electron originating from the electron transport chain (Fig 2) react with the iron(III)-hydroxyl intermediate to form iron(II)-water. At this point, the enzyme-substrate complex dissociates with the release of the product (Oleoyl-CoA) and three water molecules.

Biological Significance

A mutation in one of these nine histidines causes the enzyme to become nonfunctional.^[2] . The inactivation of SCD1 also has been known to inhibit cancer cell growth ^[4] . The inactivation of SCD1 is also commonly caused by a frameshift mutation by the addition of a proline at the 279th position. In the wild type SCD1 protein, this position contains an . A ‘CCC’ codon is inserted into the 5th exon at the 835 position SCD1 gene. This mutation results in a loss of function of SCD1. This study was done using a mouse model. In mice with this mutation, hair loss, similar to alopecia, occurs. The mice were also found to be lean during their lifespan due to decreased triglyceride synthesis connected to the loss of SCD1 function ^[9]