presqualene-pyrophosphate and farnesyl-pyrophosphate

Squalene (SQ) is a key intermediate in hopanoid biosynthesis. Many bacteria synthesize SQ from farnesyl diphosphate (FPP) in three steps: FPP to (1R,2R,3R)-presqualene diphosphate (PSPP), (1R,2R,3R)-PSPP to hydroxysqualene (HSQ), and HSQ to SQ. Chemical, biochemical, and spectroscopic methods were used to establish that HSQ synthase synthesizes (S)-HSQ. In contrast, eukaryotic squalene synthase catalyzes solvolysis of (1R,2R,3R)-PSPP to give (R)-HSQ. The bacterial enzyme that reduces HSQ to SQ does not accept (R)-HSQ as a substrate.

Topics: Catalysis; Farnesyl-Diphosphate Farnesyltransferase; Lipogenesis; Molecular Structure; Polyisoprenyl Phosphates; Sesquiterpenes; Squalene; Stereoisomerism; Triterpenes

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.

Topics: Biocatalysis; Catalytic Domain; Cations, Divalent; Crystallography, X-Ray; Escherichia coli; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression; Humans; Magnesium; Manganese; Mutagenesis, Site-Directed; NADP; Polyisoprenyl Phosphates; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Sesquiterpenes; Squalene; Static Electricity

Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation-reduction sequence of (+/-)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldimethylsilyl ethers. S(N)2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(alpha-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC(50) 1.17 +/- 0.08 muM in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(alpha-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase.

Topics: Aziridines; Catalysis; Farnesyl-Diphosphate Farnesyltransferase; Kinetics; Molecular Structure; Polyisoprenyl Phosphates; Sesquiterpenes; Squalene; Stereoisomerism

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent reduction of PSPP to squalene (SQ). These reactions are the first committed steps in cholesterol biosynthesis. When recombinant SQase was incubated with FPP in the presence of dihydroNADPH (NADPH3, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed: dehydrosqualene (DSQ), a C30 analogue of phytoene; 10(S)-hydroxysqualene (HSQ), a hydroxy analogue of squalene; and rillingol (ROH), a cyclopropylcarbinyl alcohol formed by addition of water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement of PSPP to SQ (Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77). The structure and absolute stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two independent routes. Isolation of ROH from the enzyme-catalyzed reaction provides strong evidence for a cyclopropylcarbinyl-cyclopropylcarbinyl rearrangement in the biosynthesis of squalene. By comparing the SQase-catalyzed solvolysis of PSPP in the absence of NADPH3 to the reaction in the presence of NADPH3, it is apparent that the binding of the cofactor analogue substantially enhances the ability of SQase to control the regio- and stereochemistry of the rearrangements of PSPP.

Topics: Amino Acid Sequence; Conserved Sequence; Farnesyl-Diphosphate Farnesyltransferase; Models, Chemical; Molecular Sequence Data; NADP; Polyisoprenyl Phosphates; Protein Conformation; Recombinant Proteins; Sesquiterpenes; Squalene; Stereoisomerism

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.

Topics: Binding, Competitive; Carbon Radioisotopes; Farnesyl-Diphosphate Farnesyltransferase; Hydrogen; Kinetics; Models, Chemical; NADP; Polyisoprenyl Phosphates; Protein Binding; Saccharomyces cerevisiae; Sesquiterpenes; Squalene; Substrate Specificity; Tritium

Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent reductive rearrangement of PSPP to squalene. Previous studies of the mechanism of addition of FPP to the enzyme have led to conflicting interpretations of initial velocity measurements (Beytia, E., Qureshi, A. A., and Porter, J.W. (1973) J. Biol. Chem. 248, 1856-1867; Agnew, W.S., and Popjak, G. (1978) J. Biol. Chem. 253, 4566-4573). Initial velocities for synthesis of PSPP and squalene were measured over a wider range of FPP and NADPH concentrations than previously reported, using a soluble form of recombinant enzyme. In the absence of NADPH, PSPP formation was activated by FPP at concentrations above approximately 0.5 microM. At fixed levels of NADPH, the dependence of initial rates of PSPP and squalene synthesis on FPP concentrations indicated that the C15 substrate added by a sequential mechanism. In addition, NADPH stimulated synthesis of PSPP by 40-fold at saturating levels of the cofactor. This stimulation is, at least in part, by reduction of PSPP to squalene.

Topics: Enzyme Activation; Farnesyl-Diphosphate Farnesyltransferase; Kinetics; Mathematics; Models, Theoretical; NADP; Oxidation-Reduction; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sesquiterpenes; Squalene

Squalene synthetase has been purified to homogeneity from yeast. It is a single polypeptide of Mr 47,000. This enzyme catalyzes the synthesis of squalene from farnesyl diphosphate via presqualene diphosphate. In the presence of reduced pyridine nucleotides, presqualene diphosphate and squalene are produced in a ratio of 6:1 from either the purified protein or the crude microsomal fraction.

Topics: Chromatography; Electrophoresis, Polyacrylamide Gel; Farnesyl-Diphosphate Farnesyltransferase; Isoelectric Focusing; Molecular Weight; NAD; NADP; Oxidoreductases; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sesquiterpenes; Squalene

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.

Topics: Chromatography, DEAE-Cellulose; Farnesyl-Diphosphate Farnesyltransferase; Glucosides; Isoelectric Focusing; Molecular Weight; Oxidoreductases; Polidocanol; Polyethylene Glycols; Polyisoprenyl Phosphates; Sesquiterpenes; Solubility; Squalene

In the first part of the review the background to the discovery of the asymmetric synthesis of squalene from two molecules of farnesyl pyrophosphate and NADPH is described, then the stereochemistry of the overall reaction is summarized. The complexity of the biosynthesis of squalene by microsomal squalene synthetase demanded the existence of some intermediate(s) between farnesyl pyrophosphate and squalene. This demand was satisfied by the discovery of presqualene pyrophosphate, an optically active C30 substituted cyclopropylcarbinyl pyrophosphate, the absolute configuration of which at all three asymmetric centers of the cyclopropane ring was deduced to be R. Possible mechanisms for the biosynthesis of presqualene pyrophosphate and its reductive transformation into squalene are presented. In the second part of the review the nature of the enzyme is discussed. The question whether presqualene pyrophosphate is an obligate intermediate in the biosynthesis of squalene is examined, with the firm conclusion that it is. It is as yet uncertain whether the two half reactions of squalene synthesis, i.e. (i) 2 x farnesyl pyrophosphate leads to presqualene pyrophosphate; (ii) presqualene pyrophosphate + NADPH (NADH) leads to squalene, are catalyzed by one or two enzymes or by a large complex with two catalytic sites. Evidence is cited for the existence on the enzyme of two distinct binding sites with different affinities for the two farnesyl pyrophosphate molecules. The types of enzyme preparations available at present are described and types of experiments carried out with these are critically examined. The implications of the properties of a low molecular weight squalene synthetase solubilized with deoxycholate from microsomal membranes is discussed and a model for the enzyme in an organized membrane structure is presented.

Topics: Animals; Binding Sites; Detergents; Farnesol; Farnesyl-Diphosphate Farnesyltransferase; Kinetics; Microsomes, Liver; Models, Chemical; Models, Structural; NADP; Oxidoreductases; Phospholipids; Polyisoprenyl Phosphates; Sesquiterpenes; Solubility; Squalene; Substrate Specificity; Yeasts