Oxidosqualene cyclases (OSC) are enzymes involved in cyclization reactions of 2,3-oxidosqualene to form sterols or triterpenes. There are two major groups of sterol-producing OSC enzymes:
Cycloartenol synthase (CAS), found in all plants, which produces primarily cycloartenol Lanosterol synthase (LAS), found in all animals and fungi, and occasionally in plants, which produces primarily lanosterol Sterols and triterpenes are extremely diverse classes of natural products, particularly in plants, which often contain numerous OSC enzymes with different substrate and product specificities; common examples include lupeol synthase and beta-amyrin synthase. OSC enzymes' catalytic mechanism is similar to the prokaryotic squalene-hopene cyclase. Directed evolution and protein design have been used to identify small numbers of point mutations that alter the product specificities of OSC enzymes, most notably in altering a cycloartenol synthase to produce predominantly lanosterol.
Structure Oxidosqualene cyclase is a monomeric enzyme. Its active site consists of a depression between two barrel domains. The active site is mostly made up of acidic amino acids in the majority of organisms. The residues in the active site make it energetically favorable for oxidosqualene to take on a more folded conformation, which closely resembles its product. This crucially sets the substrate up for the series of reactions that form the rings. Oxidosqualene is located in the cell’s microsome membranes where it can easily harvest its hydrophobic substrate and turn out its hydrophobic product.
Function Oxidosqualene cyclases produce a wide range of sterols and triterpenes. In animals and fungi, the type known as Lanosterol synthase produces lanosterol, which is then converted through many steps into cholesterol. Products produced by other types of the enzyme include cycloartenol, lupeol, beta-amyrin, and many more.
Regulation
The enzyme’s genetic expression in animals is regulated by sterol regulatory element binding protein (SREBP-2), a molecule which also regulates the expression of other enzymes in the cholesterol biosynthesis pathway. The SREBP-2 transcription factor increases enzymatic activity upstream of OSC, such as that of HMG-CoA reductase and squalene synthase, to increase flux through sterol synthesis process.
Mechanism
Mechanistically, the enzyme oxidosqualene:lanosterol cyclase catalyzes the formation of four rings along the long chain of the substrate (oxidosqualene), producing lanosterol. This cyclization is one of the most complex known enzyme functions and is highly selective. The reaction can be characterized by two main mechanistic series: an epoxide ring opening followed by a series of ring closures and a series of 1,2-hydride and 1,2-methyl shifts. These mechanistic steps are catalyzed by a collection of amino acids in the active site.
Ring closure In the enzyme’s active site, a histidine residue activates an aspartic acid residue (which has been identified as Asp455), which protonates the substrate’s epoxide, setting off a series of carbon-carbon bond formations that form rings. More specifically, this ring closure occurs ring by ring (labeled A-D in the mechanism below). It is widely accepted that the epoxide protonation is concurrent with the A ring closure. The cation is then transferred from C10 to C8 to form the B ring. It is the C ring formation that puzzles scholars. However, recent studies have come to a conclusion that the ring is first closed via a Markovnikov addition, forming a 5-carbon intermediate. Following this, the D ring is formed, pushing out the C ring to its 6-carbon final state. To note, QM/MM studies have shown that the protonation of this epoxide ring is the rate limiting step of the entire mechanism, indicating it may be the most regulated.
Hydride and methyl shifts The intermediate formed after the four ring closures undergoes two 1,2-hydride shifts and two 1,2-methyl shifts to be in position for final de-protonation. Molecular dynamics simulations have proved critical to understanding the conformational changes that oxidosqualene-lanosterol intermediates undergo. Researchers have identified Phe696 as a critical amino acid for controlled and specific hydride shifting in this mechanism (steps 5-6), as it speeds up the shift by lowering the energy barrier of the respective transition states.
Final de-protonation Finally, the enzyme de-protonates to yield lanosterol, which has a hydroxyl group instead of an epoxide. This hydroxyl group can be seen in the image above. The literature has shown that two amino acids in OSC are critical for this de-protonation to occur correctly and uniformly. Tyrosine at the 503rd position works in tandem with histidine at the 203rd position to de-protonate the hydrogen at the carbon adjacent to the carbocation in intermediate 8. This is accomplished as shown in the proposed mechanism below. The crystal structure of the OSC-lanosterol complex confirms that Tyr503 and His232 are in optimal positions for this final de-protonation step.
Disease relevance
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