Functional Overview
Figure 1. Esterification reaction of oleoyl-CoA and cholesterol catalyzed by SOAT. Sterol O-acyltransferase(SOAT), otherwise known as Acyl-coenzyme A:cholesterol acyltransferase(ACAT), is the first discovered member of the membrane-bound O-acyl
transferase or MBOAT enzyme group. MBOAT enzymes are responsible for the transfer of
acyl chains onto multiple types of substrates within the cell. There are 11 MBOAT enzyme types that can be found in humans, all of which serve a different function in the overall makeup of human biology.
[1]
SOAT specifically catalyzes the esterification of cholesterol for efficient storage within the cell. As a membrane lipid,cholesterol is responsible for controlling the fluidity and integrity of the membrane, along with other important biological processes. When there are high concentrations of cholesterol in the cell, cholesteryl esters can be formed for storage within the membrane.[1]
Structure
Tertiary Structure
Figure 2. Tetramer unit of SOAT shown in position within the membrane. The dimer units are identical, as indicated by the corresponding green and blue regions.
PBD 6P2P The biological assembly of SOAT is a or a . Functionally, the units of SOAT are identical and are stabilized by hydrophobic
van der Waals interactions between residues at the . Mutating these residues inhibits enzyme activity, suggesting that the dimer unit of SOAT is critical for enzyme function.
[1] Each dimer consists of two identical units, individually made up of nine labeled TM1 through TM9.
Figure 3. Labeled helices of SOAT within the membrane
The van der Waals interactions at the dimer interface stabilize the dimer between the TM1 helice of one monomer unit and the TM6
lumenal segment and TM9
cytosolic segment of the other monomer unit. Essential helices (TM1, TM5, TM6, and TM9) from the two monomers form the entrance tunnels and catalytic active site.
[2]
Tunnel System
An important structural element of SOAT is the tunnel system through which substrates enter and exit.
Figure 4. 2D layout of the SOAT tunnel system. The orientation of the tunnels shows the C tunnel opening to the cytosol and the L tunnel opening to the lumen. The T tunnel opens into the membrane, but is not quite the 90 degree shown in the 2D image.
There are 3 main tunnels in each monomer: the cytosolic (C) tunnel opening to the cytosol, the transmembrane(T) tunnel opening to the membrane, and the lumenal (L) tunnel opens to the lumen.
[2] The C tunnel opens to the cytosol of the cell and is the entrance site for the Acyl CoA substrate into the active site. Residues are involved in hydrogen bonding interactions with polar atoms on CoenzymeA to help stabilize the substrate within the binding pocket. It is important to note that the N415 residue in the PDB file provided is likely incorrect in orientation. We believe it should be flipped so that the hydrogens associated with the nitrogen atom are performing the stabilization shown. Surface representations of SOAT indicate that there are 2 alpha helices that block the entrance to the C tunnel, therefore a conformational change needs to occur to move the 2 helices so the substrate can enter the tunnel. The T tunnel opens into the membrane and is where cholesterol enters to have access to the active site. The two substrates are catalyzed by the H460 in the active site to form the cholesteryl ester. The products then leave via different pathways. The CoA-SH in the C tunnel leaves via that tunnel and is released back into the cytosol. The cholesteryl ester then leaves via either the T tunnel into the membrane or through the L tunnel into the lumen of the cell.
[2]
Active Site
Within the binding pocket, there are several . Their high preservation suggests that the local environment of the binding pocket plays a major role in SOAT activity, but their specific interactions are currently not well studied. However, have been identified as key catalytic residues.[1] Histidine, commonly used as the catalytic base to initiate acyl transferase reactions, is assumed to be the most important catalytic residue in SOAT.[3] This was confirmed as mutating H460 to alanine completely abolished enzymatic activity.[4] Additionally, H460 is highly conserved across a variety of species, further emphasizing its importance in SOAT catalysis.[1] It was hypothesized that N421 is responsible for stabilizing the transition state via an oxyanion hole with coenzymeA.[2] Additionally, mutations of W420 to alanine rendered SOAT nonfunctional, indicating that it must be essential for catalytic activity. However, its role in the mechanism was not explicitly hypothesized. We believe that it plays a role in substrate binding through with coenzyme A.
Catalytic Mechanism
The substrate of interest, , is shown bound to SOAT to visualize the binding pocket. It must be noted that cholesterol, the other substrate involved, was never correctly imaged in the active site of SOAT. Upon binding of oleoyl-CoA and cholesterol to the SOAT active site, the distal-most nitrogen on H460 acts as a base catalyst to deprotonate the hydroxyl group of cholesterol. This leaves the cholesterol oxygen with a negative charge, making it a good
nucleophile. The nucleophilic oxygen then attacks oleoyl-CoA at its carbonyl carbon, kicking electron density up to the carbonyl oxygen. The transition state is stabilized by and the newly protonated H460.
Figure 5. Mechanism for the esterification reaction of SOAT with arrow pushing.
From the transition state, excess electron density on the carbonyl oxygen is collapsed back into a double bond. This causes the bond between the carbonyl carbon and sulfur to break, shifting electron density to the sulfur atom. To complete the mechanism, the negatively charged sulfur would reclaim the hydrogen from protonated H460. CoenzymeA would exit the active site as a leaving group, leaving its oleoyl group attached to cholesterol in the form of a cholesterol ester.
It should be noted that this mechanism is largely hypothesized based on our understanding of esterification reactions and the residues involved. Further analysis is needed to confirm the proposed steps.
Inhibitors
SOAT activity is inhibited by CI-976, depending on the concentration and exposure of the inhibitor to the SOAT enzyme. When exposed, CI-976 locks itself in the of the enzyme. The can be found sandwiched between the catalytic residues H460, W420, and N421. The interactions with these residues as well as the location of the trimethoxyphenol head indicate that CI-976 inhibits the SOAT enzyme in a competitive manner by preventing the substrates from entering the catalytic center via the tunnel system. Similar to the interactions with the substrates, mutating those key catalytic residues, N421A, H460A, and H460N, result in a smaller effect of the inhibitor on the thermostability of the enzyme. [1]
Biological Relevance
SOAT has gained interest when looking at its biological relevance because it has the ability to use a wide variety of sterols in it’s mechanistic activity. The wide variety of substrates has led to SOAT being focused on as a potential drug target for many different diseases. Alzheimer’s disease, atherosclerosis, and several types of cancers have show success in treatments when targeting the SOAT enzyme’s catalytic mechanism. [1] In general, targeting SOAT could be an effective means for treating various diseases. Aberrant quantities of cholesteryl esters seem to hinder various cellular processes; thus, inhibiting SOAT expression and functionality could help reduce these adverse effects. Overall, SOAT plays an important role in cholesterol homeostasis and future research of this enzyme could lead to the discovery of therapeutic treatments for different illnesses.
Alzheimer's Disease
Alzheimer’s disease (AD) is a progressive disease that severely hinders a person’s memory and other cognitive functions. AD is the result of a significant increase in beta-amyloid (Aβ) peptide concentration. [5] Previous studies have found that the amount and distribution of intracellular cholesterol plays an important role in regulating Aβ production.[6] Therefore, SOAT inhibition could be an effective therapy for treating AD because it would reduce cholesteryl ester formation in the brain and help lower Aβ generation as well. [5] [6]
Atherosclerosis
Another disease SOAT inhibition could help treat is atherosclerosis. Buildup of cholesteryl esters from SOAT catalysis has been shown to be partially responsible for foam cell formation, one of the major indicators of atherosclerosis. Consequently, SOAT inhibitors have been studied as potential drug targets for this disease.[7]
Cancer
Increased expression of SOAT and abnormal accumulation of cholesteryl esters has also been found in multiple cancers including ovarian cancer. Therefore, inhibiting SOAT and exhausting cholesteryl ester concentrations has shown to have anti-tumor effects in terms of monitoring apoptosis, cell proliferation, and migration and invasion properties. Therapies that target SOAT regulation and expression levels could thus lead to potential treatments for ovarian and other types of cancer.[8]
