Squalene synthase: steady-state, pre-steady-state, and isotope-trapping studies

Squalene synthase catalyzes two consecutive reactions in sterol biosynthesis-the condensation of two molecules of farnesyl diphosphate (FPP) to form the cyclopropylcarbinyl intermediate presqualene diphosphate (PSPP) and the subsequent rearrangement and reduction of PSPP to form squalene. Steady-state and pre-steady-state kinetic studies, in combination with isotope-trapping experiments of enzyme.substrate complexes, indicate that two molecules of FPP add to the enzyme before NADPH and that PSPP is converted directly to squalene without dissociating from the enzyme under normal catalytic conditions. In addition, formation of PSPP or a prior conformational change in squalene synthase is the rate-limiting step for synthesis of squalene from FPP via PSPP in the presence of NADPH and for synthesis of PSPP in the absence of NADPH. Squalene synthase is inhibited at high concentrations of FPP. Inhibition is specific for the formation of squalene, but not PSPP, and is competitive with respect to NADPH. In addition, the binding of either NADPH or a third, nonreacting molecule of FPP stimulates the rate of PSPP formation. A kinetic mechanism is proposed to account for these observations.     

Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal

Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.     

Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1

Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His(6) tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His(6)-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [(14)C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg(2+) for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K(m) of FPP is 0.97 +/- 0.10 microM and the k(cat) is 1.74 +/- 0.04 s(-1). Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 +/- 13.6 nM.     

Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase

Saccharomyces cerevisiae utilizes several regulatory mechanisms to maintain tight control over the intracellular level of farnesyl diphosphate (FPP), the central precursor to nearly all yeast isoprenoid products. High-level production of non-native isoprenoid products requires that FPP flux be diverted from production of sterols to the heterologous metabolic reactions. To do so, expression of the gene encoding squalene synthase (ERG9), the first committed step in sterol biosynthesis, was down-regulated by replacing its native promoter with the methionine-repressible MET3 promoter. The intracellular levels of FPP were then assayed by expressing the gene encoding amorphadiene synthase (ADS) and converting the FPP to amorphadiene. Under certain culture conditions amorphadiene production increased fivefold upon ERG9 repression. With increasing flux to amorphadiene, squalene and ergosterol production each decreased. The levels of these three metabolites were dependent not only upon the level of ERG9 repression, but also the timing of its repression relative to the induction of ADS and genes responsible for enhancing flux to FPP.     

Arabidopsis thaliana contains a single gene encoding squalene synthase

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. Arabidopsis thaliana contains two SQS-annotated genomic sequences, At4g34640 (SQS1) and At4g34650 (SQS2), organized in a tandem array. Here we report that the SQS1 gene is widely expressed in all tissues throughout plant development, whereas SQS2 is primarily expressed in the vascular tissue of leaf and cotyledon petioles, and the hypocotyl of seedlings. Neither the complete A. thaliana SQS2 protein nor the chimeric SQS resulting from the replacement of the 69 C-terminal residues of SQS2 by the 111 C-terminal residues of the Schizosaccharomyces pombe SQS were able to confer ergosterol prototrophy to a Saccharomyces cerevisiae erg9 mutant strain lacking SQS activity. A soluble form of SQS2 expressed in Escherichia coli and purified was unable to synthesize SQ from FPP in the presence of NADPH and either Mg²⁺ or Mn²⁺. These results demonstrated that SQS2 has no SQS activity, so that SQS1 is the only functional SQS in A. thaliana. Mutational studies revealed that the lack of SQS activity of SQS2 cannot be exclusively attributed to the presence of an unusual Ser replacing the highly conserved Phe at position 287. Expression of green fluorescent protein (GFP)-tagged versions of SQS1 in onion epidermal cells demonstrated that SQS1 is targeted to the endoplasmic reticulum (ER) membrane and that this location is exclusively dependent on the presence of the SQS1 C-terminal hydrophobic trans-membrane domain.     

Tellurite Specifically Affects Squalene Epoxidase: Investigations Examining the Mechanism of Tellurium-Induced Neuropathy

Abstract: A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 µ M tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo( a )pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo( a )pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.     

Brain Isoprenoids Farnesyl Pyrophosphate and Geranylgeranyl Pyrophosphate are Increased in Aged Mice

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23?months) as compared to middle-aged mice (12?months) and young mice (3?months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.     

Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis

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 alpha 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.     

Cloning, expression and characterization of squalene synthase from Inonotus obliquus

The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.     

Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.     

Selective Inhibition of Cholesterol Biosynthesis in Brain Cells by Squalestatin 1

Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC₅₀ = 37 n M ), pig brain (IC₅₀ = 21 n M ), and C6 glioma cells (IC₅₀ = 35 n M ) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis -isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [³H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21 ^ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [³H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC₅₀ = 3–5 µ M ) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F-P-P.     

In silico analysis of squalene synthase in Fabaceae family using bioinformatics tools

Triterpenoid saponins are a diverse group of bioactive compounds, which are used for possessing of many biomedical and pharmaceutical products. Generally, squalene synthase (SQS) is defined as an emerging and essential branch point enzyme far from the major pathway of isoprenoids biosynthetic and a latent adjusting point, which manages carbon flux into triterpenes biosynthesis and sterols. The present study deals with the detailed characterization of SQS by bioinformatics approaches to evaluate physicochemical properties, structural characteristics including secondary and 3D structure prediction and functional analysis from eight plants related to Fabaceae family and Arabidopsis thaliana. Bioinformatics analysis revealed that SQS proteins have two transmembrane regions in the C-terminal. The predicted motifs were used to design universal degenerate primers for PCR analysis and other molecular applications. Phylogenetic analysis showed conserved regions at different stretches with maximum homology in amino acid residues within all SQSs. The secondary structure prediction results showed that the amino acid sequence of all squalene synthases had α helix and random coil as the main components. The reliability of the received model was confirmed using the ProSA and RAMPAGE programs. Determining of active site by CASTp proposes the possibility of using this protein as probable medication target. The findings of the present study may be useful for further assessments on characterization and cloning of squalene synthase.     

Characterization of botryococcene synthase enzyme activity, a squalene synthase-like activity from the green microalga Botryococcus braunii, Race B

The extracellular matrix of the alga Botryococcus braunii, Race B, consists mainly of botryococcenes, which have potential as a hydrocarbon fuel. Botryococcenes are structurally similar to squalene raising the possibility of a common enzyme for the biosynthesis of both. While B. braunii squalene synthase (SS) enzyme activity has been documented, botryococcene synthase (BS) enzyme activity has not been. In the current study, an assay for BS activity has been developed and used to show that many of the assay conditions for BS enzyme activity are similar to those of SS. However, SS enzyme activity is stimulated by Tween 80 while BS enzyme activity is inhibited. Moreover, BS enzyme activity was correlated with the accumulation of botryococcenes during a B. braunii culture growth cycle, which was distinctly different from the profile of SS enzyme activity. While the current results indicate a conservation of enzymological features amongst the BS and SS enzymes, raising the possibility of one enzyme capable of catalyzing both activities, they are also consistent with these two activities arising from separate and distinct enzymes.     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

油茶种仁鲨烯合酶基因的分子特性与表达分析

角鲨烯含量是高品质植物食用油评判标准的重要指标之一。鲨烯合酶是角鲨烯合成直接相关的上游调控关键酶。本研究在已构建的油茶种仁转录组数据库基础上,设计特异引物,采用RACE技术获得油茶鲨烯合酶基因的全长cDNA序列,命名为CoSQS(Gen Bank登录号为JX914592)。生物信息学分析结果表明:该序列全长1554 bp,其中含全长的开放阅读框为1245 bp,编码415个氨基酸残基,CoSQS蛋白为弱碱性非分泌型蛋白,具有2个明显的跨膜区和2个角鲨烯和番茄红素合成酶的特异信号区。与柿Dk SQS同源蛋白亲缘关系最近,属于疏水性蛋白。亚细胞定位试验结果显示该基因定位于叶绿体。实时荧光定量PCR分析表明,5-10月间,油茶种仁中CoSQS基因表达量呈现先上升后下降的趋势,转录最高峰在9月下旬。通过关联分析表明CoSQS基因表达量与角鲨烯含量密切相关。     

Identification and optimization of tetrahydro-2H-3-benzazepin-2-ones as squalene synthase inhibitors

Novel squalene synthase inhibitors are disclosed. SAR and pharmacological profile of selected compounds are discussed.     

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N -glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae , we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis -prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2 , only HMG2 overexpression was able to restore growth of the yta7 Δ cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.