Squalene synthase (SQS) or farnesyl-diphosphate:farnesyl-diphosphate farnesyl transferase is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
localized to the membrane of the
endoplasmic reticulum. SQS participates in the
isoprenoid biosynthetic pathway, catalyzing a two-step reaction in which two identical molecules of
farnesyl pyrophosphate (FPP) are converted into
squalene, with the consumption of
NADPH.
Catalysis
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
by SQS is the first committed step in
sterol synthesis, since the squalene produced is converted exclusively into various sterols, such as
cholesterol
Cholesterol is any of a class of certain organic molecules called lipids. It is a sterol (or modified steroid), a type of lipid. Cholesterol is biosynthesized by all animal cells and is an essential structural component of animal cell memb ...
, via a complex, multi-step pathway. SQS belongs to
squalene/phytoene synthase family
The squalene/phytoene synthase family represents proteins that catalyze the head-to-head condensation of C15 and C20 prenyl units (i.e. farnesyl diphosphate and genranylgeranyl diphosphate). This enzymatic step constitutes part of steroid and caro ...
of proteins.
Diversity
Squalene synthase has been characterized in animals, plants, and yeast.
In terms of structure and mechanics, squalene synthase closely resembles
phytoene synthase (PHS), another
prenyltransferase. PHS serves a similar role to SQS in plants and bacteria, catalyzing the synthesis of
phytoene, a precursor of
carotenoid compounds.
Structure
Squalene synthase (SQS) is localized exclusively to the
membrane of the
endoplasmic reticulum (ER).
SQS is anchored to the membrane by a short
C-terminal membrane-spanning domain.
The
N-terminal catalytic domain of the enzyme protrudes into the
cytosol, where the soluble
substrates are bound.
Mammalian forms of SQS are approximately 47
kDa and consist of ~416
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s. The
crystal structure
In crystallography, crystal structure is a description of the ordered arrangement of atoms, ions or molecules in a crystalline material. Ordered structures occur from the intrinsic nature of the constituent particles to form symmetric pattern ...
of human SQS was determined in 2000, and revealed that the protein was composed entirely of
α-helices. The enzyme is folded into a single
domain
Domain may refer to:
Mathematics
*Domain of a function, the set of input values for which the (total) function is defined
** Domain of definition of a partial function
** Natural domain of a partial function
**Domain of holomorphy of a function
* ...
, characterized by a large central channel. The
active sites of both of the two half-reactions catalyzed by SQS are located within this channel. One end of the channel is open to the cytosol, whereas the other end forms a
hydrophobic
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, ...
pocket.
SQS contains two conserved
aspartate-rich sequences, which are believed to participate directly in the catalytic mechanism.
These aspartate-rich motifs are one of several conserved structural features in class I isoprenoid biosynthetic enzymes, although these enzymes do not share sequence
homology.
Mechanism
Squalene synthase (SQS) catalyzes the reductive dimerization of farnesyl pyrophosphate (FPP), in which two identical molecules of FPP are converted into one molecule of squalene. The reaction occurs in two steps, proceeding through the intermediate presqualene pyrophosphate (PSPP). FPP is a
soluble allylic compound containing 15 carbon atoms (C
15), whereas squalene is an insoluble, C
30 isoprenoid.
This reaction is a head-to-head
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ...
synthesis, because the two FPP molecules are both joined at the C4 position and form a 1-1' linkage. This stands in contrast to the 1'-4 linkages that are much more common in isoprene biosynthesis than 4-4' linkages.
The reaction mechanism of SQS requires a
divalent cation, often Mg
2+, to facilitate binding of the
pyrophosphate groups on FPP.
FPP condensation
In the first half-reaction, two identical molecules of farnesyl pyrophosphate (FPP) are bound to squalene synthase (SQS) in a sequential manner. The FPP molecules bind to distinct regions of the enzyme, and with different binding affinities.
Starting at the top of the catalytic cycle below, the reaction begins with the ionization of FPP to generate an allylic
carbocation. A
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
residue (Tyr-171) plays a critical role in this step by serving as a proton donor to facilitate abstraction of pyrophosphate. Moreover, the resulting phenolate anion can stabilize the resulting carbocation through
cation-π interactions, which would be particularly strong due to the highly electron-rich nature of the phenolate anion. The allylic cation generated is then attacked by the olefin of a second molecule of FPP, affording a tertiary carbocation. The phenolate anion generated previously then serves as a base to abstract a proton from this adduct to form a cyclopropane product, presqualene pyrophosphate (PSPP). The PSPP created remains associated with SQS for the second reaction.
The importance of a tyrosine residue in this process was demonstrated by mutagenesis studies with rat SQS (rSQS),
and by the fact that Tyr-171 is conserved in all known SQSs (and
PHSs).
In rSQS, Tyr-171 was converted to aromatic residues
Phe and
Trp, as well as hydroxyl-containing residue
Ser. None of these mutants were able to convert FPP to PSPP or squalene, demonstrating that aromatic rings or alcohols alone are insufficient for converting FPP to PSPP.
PSPP rearrangement and reduction
In the second half-reaction of SQS, presqualene pyrophosphate (PSPP) moves to a second reaction site within SQS. Keeping PSPP in the central channel of SQS is thought to protect the reactive intermediate from reacting with water.
From PSPP, squalene is formed by a series of carbocation rearrangements. The process begins with ionization of pyrophosphate, giving a cyclopropylcarbinyl cation. The cation rearranges by a
1,2-migration of a cyclopropane C–C bond to the carbocation, forming the bond shown in blue to give a cyclobutyl carbocation. Subsequently, a second 1,2-migration occurs to form another cyclopropylcarbinyl cation, with the cation resting on a tertiary carbon. This resulting carbocation is then ring-opened by a hydride delivered by
NADPH, giving squalene, which is then released by SQS into the membrane of the
endoplasmic reticulum.
While cyclopropylcarbinyl-cyclopropylcarbinyl rearrangements can proceed through discrete cyclobutyl cation intermediates, the supposed cyclobutyl cation could not be trapped in model studies. Thus, the cyclobutyl cation may actually be a
transition state
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked ...
between the two cyclopropylcarbinyl cations, rather than a discrete intermediate. The stereochemistry of the intermediates and the olefin geometry in the final product is dictated by the
suprafacial nature of the 1,2-shifts and
stereoelectronic requirements. While other mechanisms have been proposed, the mechanism shown above is supported by isolation of rillingol, which is the alcohol formed from trapping the second cyclopropylcarbinyl cation with water.
Regulation
FPP is an important metabolic intermediate in the
mevalonate pathway that represents a major branch point in
terpenoid pathways.
FPP is used to form several important classes of compounds in addition to sterols (''via'' squalene), including
ubiquinone and
dolichols. SQS catalyzes the first committed step in sterol biosynthesis from FPP, and is therefore important for controlling the flux towards
sterol vs. non-sterol products. The activity of SQS is intimately related to the activity of
HMG-CoA reductase, which catalyzes the rate-limiting step of the mevalonate pathway. High levels of
LDL-derived
cholesterol
Cholesterol is any of a class of certain organic molecules called lipids. It is a sterol (or modified steroid), a type of lipid. Cholesterol is biosynthesized by all animal cells and is an essential structural component of animal cell memb ...
inhibit HMG-CoA reductase activity significantly, since mevalonate is no longer needed for sterol production. However, residual HMG-CoA reductase activity is observed even with very high LDL levels, such that FPP can be made for forming non-sterol products essential for cell growth.
To prevent this residual FPP from being used for sterol synthesis when sterols are abundant, SQS activity declines significantly when LDL levels are high. This suppression of SQS activity is better thought of as a flux control mechanism, rather than a way to regulate cholesterol levels. This is since HMG-CoA reductase is the more significant control factor for regulating cholesterol synthesis (its activity is 98% inhibited when LDL levels are high).
Regulation by sterols
SQS regulation occurs primarily at the level of SQS
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
transcription.
The
sterol regulatory element binding protein (SREBP) class of
transcription factors is central to regulating genes involved in cholesterol
homeostasis
In biology, homeostasis ( British also homoeostasis) (/hɒmɪə(ʊ)ˈsteɪsɪs/) is the state of steady internal, physical, and chemical conditions maintained by living systems. This is the condition of optimal functioning for the organism and ...
, and is important for controlling levels of SQS transcription. When sterol levels are low, an inactive form of SREBP is cleaved to form the active transcription factor, which moves to the nucleus to induce transcription of the SQS gene. Of the three known SREBP transcription factors, only SREBP-1a and SREBP-2 activate SQS gene transcription in transgenic mouse livers. In cultured
HepG2
Hep G2 (or HepG2) is a human liver cancer cell line.
Hep G2 is an immortal cell line which was derived in 1975 from the liver tissue of a 15-year-old Caucasian male from Argentina with a well-differentiated hepatocellular carcinoma. T ...
cells, SREBP-1a appears more important than SREBP-2 in controlling activation of the SQS
promoter. However, SQS promoters have been shown to respond differently to SREBP-1a and SREBP-2 in different experimental systems.
Aside from SREBPs, accessory transcription factors are needed for maximal activation of the SQS promoter. Promoter studies using
luciferase reporter gene assays revealed that the
Sp1, and NF-Y and/or
CREB transcription factors are also important for SQS promoter activation. NF-Y and/or CREB are required for SREBP-1a to fully activate the SQS promoter, although Sp1 is also needed for SREBP-2 to do so.
Interactive pathway map
Biological Function
Squalene synthase (SQS) is an enzyme participating in the isoprenoid biosynthetic pathway. SQS synthase catalyzes the branching point between sterol and nonsterol biosynthesis, and commits farnesyl pyrophosphate (FPP) exclusively to production of sterols.
An important sterol produced by this pathway is
cholesterol
Cholesterol is any of a class of certain organic molecules called lipids. It is a sterol (or modified steroid), a type of lipid. Cholesterol is biosynthesized by all animal cells and is an essential structural component of animal cell memb ...
, which is used in cell membranes and for the synthesis of
hormone
A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are required ...
s.
SQS competes with several other enzymes for use of FPP, since it is a precursor for a variety of terpenoids. Decreases in SQS activity limit flux of FPP to the sterol pathway, and increase the production of nonsterol products. Important nonsterol products include
ubiquinone,
dolichols,
heme A, and farnesylated proteins
Development of squalene synthase
knockout mice has demonstrated that loss of squalene synthase is lethal, and that the enzyme is essential for development of the
central nervous system
The central nervous system (CNS) is the part of the nervous system consisting primarily of the brain and spinal cord. The CNS is so named because the brain integrates the received information and coordinates and influences the activity of all p ...
.
Disease Relevance
Squalene synthase is a target for the regulation of cholesterol levels. Increased
expression of SQS has been shown to elevate cholesterol levels in mice.
Therefore,
inhibitors of SQS are of great interest in the treatment of
hypercholesterolemia and prevention of
coronary heart disease (CHD).
It has also been suggested that variants in this enzyme may be part of a genetic association with hypercholesterolemia.
Squalene synthase inhibitors
Squalene synthase inhibitors have been shown to decrease cholesterol synthesis, as well as to decrease plasma
triglyceride
A triglyceride (TG, triacylglycerol, TAG, or triacylglyceride) is an ester derived from glycerol and three fatty acids (from ''tri-'' and ''glyceride'').
Triglycerides are the main constituents of body fat in humans and other vertebrates, as ...
levels.
SQS inhibitors may provide an alternative to
HMG-CoA reductase inhibitors
Statins, also known as HMG-CoA reductase inhibitors, are a class of lipid-lowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. They are the most common cholesterol-lowering drugs.
Low ...
(statins), which have problematic side effects for some patients.
Squalene synthase inhibitors that have been investigated for use in the prevention of
cardiovascular disease include
lapaquistat (TAK-475),
zaragozic acid, and RPR 107393.
Despite reaching phase II
clinical trial
Clinical trials are prospective biomedical or behavioral research studies on human participants designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel vaccines, drugs, diet ...
s, lapaquistat was discontinued by 2008.
Squalene synthase homolog inhibition in ''
Staphylococcus aureus'' is currently being investigated as a virulence factor-based
antibacterial
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention ...
therapy.
Model organisms
Model organisms have been used in the study of FDFT1 function. A conditional
knockout mouse
A knockout mouse, or knock-out mouse, is a genetically modified mouse (''Mus musculus'') in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are importan ...
line called ''Fdft1
tm1a(KOMP)Wtsi'' was generated at the
Wellcome Trust Sanger Institute.
Male and female animals underwent a standardized
phenotypic screen
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological prop ...
to determine the effects of deletion.
Additional screens performed: - In-depth immunological phenotyping
''
''
References
External links
*
{{Alkyl and aryl transferases
Biosynthesis
EC 2.5.1