Squalene synthetase

Popj�k, George; Agnew, William S.

doi:10.1007/bf00218354

Cited by 44 publications

(31 citation statements)

References 54 publications

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“…Both FPP and GGPP are positioned at branch-points leading to the synthesis of longer-chain isoprenoids. Head-to-head condensation of two FPP units, catalyzed by squalene synthase (SQS), leads to the first precursor in the sterol pathway [2]. In addition, FPP is used for the synthesis of dolichols, ubiquinone and heme A. FPP and GGPP also serve substrates for protein isoprenylation reactions [3].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Holstein

Tong

Kuder

et al. 2009

Lipids

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Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.

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Section: Introductionmentioning

confidence: 99%

Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Holstein

Tong

Kuder

et al. 2009

Lipids

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“…SS catalyzes the condensation of two molecules of farnesyl diphosphate to form the linear 30 carbon compound squalene (Popjak et al, 1961a(Popjak et al, , 1961b, and this activity has been localized to the smooth endoplasmic reticulum (ER; Popjak and Agnew, 1979;Stamellos et al, 1993). Additional studies have suggested that the carboxy-terminal portion of the SS protein anchors the protein to the ER membrane, whereas the catalytic site of the enzyme is associated with the amino-terminal portion of the protein found on the cytoplasmic face of the ER (Robinson et al, 1993).…”

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confidence: 99%

Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco

Devarenne¹,

Ghosh

Chappell³

2002

Plant Physiology

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Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

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“…It has also been suggested that the two FPP molecules bind sequentially with the donor FPP binding first (9,12) and that translocation of PSQPP from the first to the second reaction center occurs without its release from the enzyme (12,13). Several residues thought to be involved in the SQS catalytic machinery have been identified through site-specific mutagenesis (14).…”

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confidence: 99%

Crystal Structure of Human Squalene Synthase

Pandit¹,

Danley²,

Schulte³

et al. 2000

Journal of Biological Chemistry

216

135

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Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two farnesyl diphosphate (FPP) molecules. The reaction is unique when compared with those of other FPP-utilizing enzymes and proceeds in two distinct steps, both of which involve the formation of carbocationic reaction intermediates. Because FPP is located at the final branch point in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of squalene synthase represents the first committed step in the formation of cholesterol, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structures of recombinant human squalene synthase complexed with several different inhibitors. The structure shows that SQS is folded as a single domain, with a large channel in the middle of one face. The active sites of the two half-reactions catalyzed by the enzyme are located in the central channel, which is lined on both sides by conserved aspartate and arginine residues, which are known from mutagenesis experiments to be involved in FPP binding. One end of this channel is exposed to solvent, whereas the other end leads to a completely enclosed pocket surrounded by conserved hydrophobic residues. These observations, along with mutagenesis data identifying residues that affect substrate binding and activity, suggest that two molecules of FPP bind at one end of the channel, where the active center of the first half-reaction is located, and then the stable reaction intermediate moves into the deep pocket, where it is sequestered from solvent and the second half-reaction occurs. Five ␣ helices surrounding the active center are structurally homologous to the active core in the three other isoprenoid biosynthetic enzymes whose crystal structures are known, even though there is no detectable sequence homology.The isoprenoid biosynthetic pathway yields a structurally diverse family of low molecular weight molecules with a variety of physiological functions (1-3). In humans, the pathway produces such critical end-products as cholesterol, bile acids, dolichol, ubiquinone, steroid hormones, and prenylated proteins. Chain length-selective prenyltransferases catalyze the successive head-to-tail addition of the 5-carbon (C5-) isopentenyl diphosphate to the growing isoprene chain to form a series of linear C10-, C15-, C20-and C25-isoprenoid diphosphates. Cyclic terpenes are formed from these linear isoprenoid diphosphates through the actions of numerous class I terpene cyclases (3). The longer 30-carbon linear terpene, squalene, of cholesterol biosynthesis and the 40-carbon linear terpene, phytoene, of carotenoid biosynthesis are formed by head-to-head condensations, respectively, of two 15-carbon and 20-carbon isoprenoid diphosphates, through the actions of the prenyltransferases squalene synthase and phytoene synthase (4) and are subsequently converted into their respective cyclic terpenes through the action of specific class II terpene c...

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Squalene synthetase

Cited by 44 publications

References 54 publications

Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco

Crystal Structure of Human Squalene Synthase

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