Publications

• Structural insights into the catalytic mechanism of human squalene synthase.

  Liu C.I., Jeng W.Y., Chang W.J., Shih M.F., Ko T.P., Wang A.H.

  Squalene synthase (SQS) is a divalent metal-ion-dependent enzyme that catalyzes the two-step reductive `head-to-head' condensation of two molecules of farnesyl pyrophosphate to form squalene using presqualene diphosphate (PSPP) as an intermediate. In this paper, the structures of human SQS and its ... >> More

  Squalene synthase (SQS) is a divalent metal-ion-dependent enzyme that catalyzes the two-step reductive `head-to-head' condensation of two molecules of farnesyl pyrophosphate to form squalene using presqualene diphosphate (PSPP) as an intermediate. In this paper, the structures of human SQS and its mutants in complex with several substrate analogues and intermediates coordinated with Mg2+ or Mn2+ are presented, which stepwise delineate the biosynthetic pathway. Extensive study of the SQS active site has identified several critical residues that are involved in binding reduced nicotinamide dinucleotide phosphate (NADPH). Based on mutagenesis data and a locally closed (JK loop-in) structure observed in the hSQS-(F288L)-PSPP complex, an NADPH-binding model is proposed for SQS. The results identified four major steps (substrate binding, condensation, intermediate formation and translocation) of the ordered sequential mechanisms involved in the `1'-1' isoprenoid biosynthetic pathway. These new findings clarify previous hypotheses based on site-directed mutagenesis and biochemical analysis. << Less

  Acta Crystallogr. D 70:231-241(2014) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase.

  Ericsson J., Appelkvist E.L., Thelin A., Chojnacki T., Dallner G.

  Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in micros ... >> More

  Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes. << Less

  J Biol Chem 267:18708-18714(1992) [PubMed] [EuropePMC]

• Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

  Shechter I., Klinger E., Rucker M.L., Engstrom R.G., Spirito J.A., Islam M.A., Boettcher B.R., Wienstein D.B.

  Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-3 ... >> More

  Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa. << Less

  J. Biol. Chem. 267:8628-8635(1992) [PubMed] [EuropePMC]

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

  Radisky E.S., Poulter C.D.

  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-s ... >> More

  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. << Less

  Biochemistry 39:1748-1760(2000) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

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

  Pandit J., Danley D.E., Schulte G.K., Mazzalupo S., Pauly T.A., Hayward C.M., Hamanaka E.S., Thompson J.F., Harwood H.J. Jr.

  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 invo ... >> More

  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. << Less

  J. Biol. Chem. 275:30610-30617(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Structure and regulation of mammalian squalene synthase.

  Tansey T.R., Shechter I.

  Mammalian squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. SQS produces squalene in an unusual two-step reaction in which two molecules of farnesyl diphosphate are condensed head-to-head. Recent st ... >> More

  Mammalian squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. SQS produces squalene in an unusual two-step reaction in which two molecules of farnesyl diphosphate are condensed head-to-head. Recent studies have advanced understanding of the reaction mechanism, the functional domains of the enzyme, and transcriptional regulation of the gene. Site-directed mutagenesis has identified conserved Asp, Tyr, and Phe residues that are essential for SQS activity. The Asp residues are hypothesized to be required for substrate binding; the Tyr and Phe residues may stabilize carbocation reaction intermediates. The elucidation of SQS crystal structure will most likely direct future research on the relationship between enzyme structure and function. SQS activity, protein, and mRNA levels are regulated by cholesterol status and by the cytokines TNF-alpha and IL-1beta. Activation of the SQS promoter in response to cholesterol deficit is mediated by sterol regulatory element binding proteins SREBP-1a and SREBP-2. The precise contributions made by individual SREBPs and accessory transcription factors to SQS transcriptional control, and the mechanisms underlying cytokine regulation of SQS are major foci of current research. << Less

  Biochim Biophys Acta 1529:49-62(2000) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase.

  LoGrasso P.V., Soltis D.A., Boettcher B.R.

  Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein. For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic p ... >> More

  Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein. For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured. A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme. Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase. The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme. In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively. Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme. << Less

  Arch Biochem Biophys 307:193-199(1993) [PubMed] [EuropePMC]