Lanosterol Synthase: The Most Effective Way to Lower Cholesterol
Created by Logan Rosenberg
The membrane-bound enzyme oxidosqualene cyclase (OSC, PDB ID: 1W6K) from Homo sapiens, commonly known as lanosterol synthase, plays a main role in forming the steroid scaffold in sterol biosynthesis (1). More specifically, lanosterol synthase catalyzes the cyclization of (3S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus (2). The critical task of OSC is to properly fold the open triterpene oxidosqualene to generate the four-membered steroid ring. Despite its structurally critical role, lanosterol synthase is not a regulatory enzyme in sterol biosynthesis, but rather indirectly plays a role in lipid regulatory processes (3). The crucial importance of oxidosqualene cyclase in cholesterol biosynthesis represents a target for the discovery of novel OSC inhibitors that function as anticholesteraemic drugs (1).
The molecular weight of lanosterol synthase is 83,294.72 Da and the isoelectric point (pI) is 6.16. The primary structure of this enzyme has 732 amino acids with several natural variations or mutations. These natural variations can be seen in amino acids 175 (Arg to Gln), 310 (His to Arg), 614 (Arg to Trp), 642 (Leu to Val), and 688 (Pro to Leu). The main catalytic amino acids are the acidic Asp 455 residue and the basic His 232 residue. The secondary structure of oxidosqualene cyclase consists of about 50% α helices, about 7% β strands, about 1% β turns, and about 42% random coils. OSC does not have a prosthetic group or any associations with metal ions (4).
The structure of oxidosqualene cyclase was deduced through x-ray crystallographic results of OSC with both the inhibitor Ro 48-8071 and its product lanosterol. These findings show that the secondary structure of lanosterol synthase consists of alpha helices, beta sheets, and random coils. Human OSC is a monomer that consists of two α-α barrel domains that are connected by loops and three smaller β-structures (1). The two domains of the single subunit form a dumbbell shape. Domain 1 is an α6-α6 barrel of two concentric rings of parallel α helices. Domain 2 is inserted into domain 1 and contains an evolved form of the α6-α6 barrel that is found in domain 1. The amino ends of the inner α helices of both barrels point toward the molecular center. This consists of long loops from both domains that form a β structure and enclose the active site cavity (5). The amino-terminal region of OSC is positively charged and thus polar. It fills the space between the two domains and is thought to stabilize their relative orientations. There are five signature QW-sequence motifs that are located toward the carboxy terminus and reside outside the two main α-α barrel domains (1). These QW motifs connect surface α helices and are thought to stabilize OSC by tightening the protein structure. This avoids structural damage that can occur from absorbing the reaction energy during the highly exergonic cyclization (5).
Human oxidosqualene cyclase is an integral membrane protein associated with the endoplasmic reticulum in eukaryotes. Since it is a monotopic membrane protein, it inserts into the membrane but does not span the bilayer. The native membrane surrounding OSC must be disrupted with detergents in order to crystallize the protein. Detergents do not cause any denaturation of lanosteral synthase (4). The detergent octyl- β –D-glucopyranoside (β–OG) is used as a ligand merely to induce crystallization. The approximate orientation of oxidosqualene cyclase is determined by aligning the glucose head groups of two bound β–OG detergent molecules in the polar membrane layer and the octyl groups parallel to the fatty-acid chains in the bilayer. The two polar headgroups of β–OG bind along one of the three helices in the membrane-binding region of OSC. The nonpolar octyl tails of the β–OG detergent molecules then interact with hydrophobic regions of neighboring OSC molecules. These results show that the region in OSC that binds to the membrane is from domain 2, and can be detected by bound detergent molecules and by its hydrophobic surface. This membrane-binding surface consists of a plateau with a diameter of 25 angstroms and has a channel that leads to the active site cavity (1).
The active site is located in the central cavity between the two α-α barrel domains, which can be determined by crystallizing OSC with the bound competitive inhibitor N,N-dimethyldodecylamine-N-oxide (LDAO). The cavity is lined by aromatic residues and is mainly hydrophobic with a polar patch at the top. It is of suitable size to bind squalene in its required conformation (5). The channel leading to the central cavity has a constriction site, or gate, that separates it from the active site cavity. The substrate (3S)-2,3-oxidosqualene can pass through this constriction site by conformational changes in the side chains of residues Tyr 237, Cys 233, and Ile 524 or by the rearrangement of the strained loops from amino acids 516-524 and 697-699 (1). When the substrate (3S)-2,3-oxidosqualene enters the central cavity through this constriction site, these unfavorable conformation changes facilitate the initiation of catalysis (5).
Squalene-hopene cyclase (SHC, PDB ID: 2SQC) from Alicyclobacillus Acidocaldarius is a notable protein that shows unmistakable structural and functional similarities to human oxidosqualene cyclase. The comparison of these two proteins was investigated through bioinformatics with the help of the Position-Specific-Iterated Basic Alignment Search Tool (PSI-BLAST) and the Dali server. PSI-BLAST is a program used to find proteins with similar primary structures to a protein query. This program assigns an E value to a subject protein that has sequence homology to the query. The E value is calculated by looking at the total sequence homology and assigning gaps. Gaps are amino acids that exist in the subject’s sequence but not in the query’s sequence. The E value is larger for sequence homologies that have larger gaps. The Dali server is a method for finding proteins with tertiary structures similar to a query. It gives tertiary structure similarities by comparing intramolecular distances and assigning a Z-score to a subject protein. A Z-score above 2 is significant and means that the proteins have similar folds.
The results from PSI-BLAST show that squalene-hopene cyclase from Alicyclobacillus Acidocaldarius has 88% query coverage, 25% identity, and an E value of 0.0, indicating that there are no gaps and the query and subject have identical primary structures. This sequence similarity dictates analogous tertiary structures, which can be supported by the high Z-score of 42.1 obtained from the Dali server (6). A main difference in the primary structures between human oxidosqualene cyclase and squalene-hopene cyclase from Alicyclobacillus Acidocaldarius is that SHC has eight QW sequence motifs whereas OSC only has five QW sequence motifs. The side chains of these QW motifs stabilize the whole protein by forming hydrogen bonds with the amino end of the adjacent outer barrel helix and with the carbonyl end of the preceding outer barrel helix. Another important difference between these primary structures is that the polar top of the active site cavity in SHC has the sequence motif DXDD (X is any amino acid) from residues 374-377 that is important for catalysis. Human oxidosqualene cyclase only has the Asp 376 residue, which is also crucial for catalysis (5). SHC catalyzes the cyclization of squalene into hopene whereas OSC catalyzes the cyclization of (3S)-2,3-oxidosqualene into lanosterol. In terms of tertiary structure differences, the OSC active site cavity is smaller than the SHC active site cavity. This results in different inhibitor conformations and explains why inhibitor design based on SHC results in inaccurate predictions for OSC (1).
The main function of human oxidosqualene cyclase is the biosynthesis of lanosterol from the substrate (3S)-2,3-oxidosqualene. Lanosterol is the tetracyclic triterpenoid from which all steroids are derived. This conversion from (3S)-2,3-oxidosqualene to lanosterol by oxidosqualene cyclase is one of the most powerful one-step constructions known in biochemistry and is often noted as one of the most complex of all enzyme-catalyzed reactions (7). Throughout this reaction, the substrate proceeds through a series of discrete, conformationally rigid, partially cyclized carbocation intermediates (8). The first step in this reaction mechanism is for the (3S)-2,3-oxidosqualene to enter the active site cavity and adopt a chair-boat-chair conformation (1). The chemical cyclization of oxidosqualene cyclase must then be initiated by electrophilic epoxide activation (9). Protonation of the epoxide ring in this conformation triggers a cascade of ring-forming reactions to the protosterol cation. A series of 1,2-hydride and 1,2-methyl group shifts lead to backbone rearrangement and then a final deprotonation step leads to the product lanosterol. Product specificity and high stereoselectivity are observed because of the prefolded chair-boat-chair conformation of the substrate, progression through rigid, partly-cyclized carbocation intermediates, and stabilization of these intermediates by cation-π interactions (1).
Human oxidosqualene cyclase was crystallized with its product lanosterol to gain a better understanding of the structural basis for the highly exergonic and stereoselective cyclization reaction. In this case, lanosterol serves as a ligand and is bound to the active site. The crystal structure obtained from this enzyme-substrate complex allowed for the 2,3-oxidosqualene intermediates to be modeled into the active site. Lanosterol fits very closely to the shape and physiochemical properties of the active site and allows researchers to observe the important catalytic residues. The crystal structure shows that the catalytic Asp 455 residue on oxidosqualene cyclase forms a hydrogen bond with the lanosterol 3-hydroxy group, forming the polar cap of the mainly hydrophobic central cavity. This finding demonstrates that the complex cyclization reaction mechanism catalyzed by lanosterol synthase is initiated when Asp 455 protonates the epoxide group of the prefolded (3S)-2,3-oxidosqualene (1). Since oxidosqualene is completely stable in neutral media, a powerful Bronsted acid is required for sufficiently strong epoxide activation. This protonation activates the Asp 455 and promotes cyclization on a shorter timescale (9). In addition, the x-ray crystallographic results show that Cys 456 and Cys 533 further increase the acidity of Asp 455 by serving as hydrogen bonding partners. After one complete catalytic cycle, Asp 455 is reprotonated either by the bulk solvent through a chain of water molecules and the carboxylate group of Glu 459, or by a proton from the final deprotonation step shifting back to Asp 455 (1).
After the catalytic Asp 455 initiates the cyclization reaction, (3S)-2,3-oxidosqualene moves through several intermediates where the four cyclic structures in lanosterol form. These four cyclic structures are labeled A-D in the order that they close. The side-chains of Phe 444, Tyr 503, and Trp 581 stabilize the intermediate cations at C-6 and C-10 after A-ring and B-ring formation by cation-π interactions. In addition,Tyr 98 serves as an important residue by pushing the methyl group at C-10 below the molecular plane. This steric hindrance produces the energetically unfavorable boat conformation of 2,3-oxidosqualene required for lanosterol B-ring formation. His 232 and Phe 696 are other important residues that play a role in C-ring formation. The side chains of these two residues are positioned in such a way that they stabilize the anti-Markovnikov secondary cation at C-14 through π-interactions. The oxidosqualene cyclase cyclization cascade stops after formation of the five-membered D ring. This is because there is no aromatic residue to stabilize the tertiary protosterol cation at C-20. Skeletal rearrangement of the protosterol C-20 cation results in the lanosterol C-8/C-9 cation. The basic His 232 residue terminates catalysis because it is the only residue close enough to accept the proton in the deprotonation of the C-8/C-9 lanosterol cation. In addition, the hydroxy group of Tyr 503 that is hydrogen bonded to His 232 is in a proper position for the final deprotonation step (1).
Oxidosqualene cyclase inhibitors serve as the basis for anticholesteraemic drugs because they hinder the synthesis of lanosterol, which is a key derivative from which all sterols are formed. Lanosterol synthase does not interfere with the synthesis of isoprenoids and coenzyme Q in the cholesterol pathway because it acts downstream of the farnesyl-pyrophosphate. Therefore, OSC inhibitors have less adverse side effects than statins, which target HMG-CoA reductase higher up in the cholesterol biosynthetic pathway (10). Although this interest in pharmaceuticals was the driving force behind searching for successful OSC inhibitors, research and experimentation has helped shed light on the precise mechanism of the enzyme. The enzymatic cyclization of (3S)-2,3-oxidosqualene can be separated into three steps, each of which can potentially be affected by an inhibitor. These three enzymatic steps are the acid-catalyzed opening of the epoxide ring, the concerted or not concerted cyclization to give a carbonium ion, and the concerted backbone rearrangement to lanosterol. A general and successful strategy to investigate novel inhibitors is to mimic the carbonium ions that are formed during A-D ring closure in the concerted cyclization reactions. This can be done by replacing the positively charged carbon atom with a nitrogen atom that is protonated at physiological pH. These inhibitors are analogues of the enzyme-bound high-energy intermediates (11). Therefore, an approach for the design of new OSC inhibitors consists of superimposing a prototype inhibitor on the high-energy intermediate of oxidosqualene in a way to optimize hydrophobic and ionic interactions with the transition state of the enzyme. The high-energy intermediate of oxidosqualene cyclization can be modeled with computer programs to stabilize cationic centers and reduce unfavorable conformations in the complex (10).
The prototype inhibitor for human oxidosqualene cyclase is Ro 48-8071 (10). The structure of Ro 48-8071 in complex with human OSC is an alternate conformation of lanosterol synthase (PDB ID: 1W6J) and provides a structural foundation for the design of new and improved inhibitors (1). The idea behind using the inhibitor Ro 48-8071 is for the amine to interact with the positive charge of the epoxide-opening region of OSC and the carbonyl of the benzophenone to interact with the negative point-charge, thus stabilizing the last protosterol cation (10). Experimentation and observation have shown that the basic nitrogen atom in Ro 48-8071 forms a charged hydrogen bond at a distance of 2.9 angstroms from the catalytic Asp 455 carboxylate that directs both oxygen atoms toward the active site cavity. The charged amino group is also stabilized by cation-π interactions with several aromatic residues. The fluorophenyl group of the benzophenone moiety is positioned between the side chains of Phe 696 and His 232. The carbonyl group on the benzophenone is hydrogen bonded to a water molecule that interacts with the backbone amide nitrogen of Ile 338. In addition, the terminal phenyl group on benzophenone interacts with the OSC residues Trp 192, Trp 130, and Phe 521. These three residues create a π-electron-rich pocket that is optimal for the stabilization of electron-deficient aromatic systems. All of these structural aspects of Ro 48-8071 dictate its function of stabilizing the last protosterol cation in lanosterol formation. Preventing the formation of lanosterol directly inhibits the biochemical synthesis of cholesterol (1).
Only recently has human oxidosqualene cyclase become one of the most promising targets for the discovery of effective anticholesteraemic drugs. Technological advances in x-ray crystallography, protein purification, and computer modeling have helped elucidate the fine details of the highly complex and powerful one-step cyclization reaction catalyzed by OSC. This enzyme can ultimately reduce the concentration of cholesterol in humans by preventing the conversion of (3S)-2,3-oxidosqualene to lanosterol with the use of bound inhibitors. Inhibition of OSC is such a promising and exciting investigation for pharmaceutical companies because it has much less adverse side effects than the widely used statins. By complementing the use of statins with OSC inhibitors, individuals can lower their cholesterol levels in a healthy way without creating additional medical complications. The importance of continued research on lanosterol synthase inhibition cannot be stressed enough and will continue to save many lives in the future.