Squalene synthetase. Inhibition by an ammonium analogue of a carbocationic intermediate in the conversion of presqualene pyrophosphate to squalene

Sandifer, Ronda M.; Thompson, Michael D.; Gaughan, R. G.; Poulter, C. Dale

doi:10.1021/ja00389a106

Cited by 48 publications

(17 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such tight binding suggested that the diphosphate moiety was involved. This conclusion was confirmed by the inhibitory effect of inorganic pyrophosphate reported below and is reinforced by studies based on the use of a transition-state analog inhibitor of squalene synthetase, which catalyzes a very similar reaction (21).…”

Section: Resultssupporting

confidence: 61%

Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

Dogbo

Laferrière

d’Harlingue

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

Phytoene is the frst C40 intermediate in the biogenesis of carotenoids. It is formed by two enzyme activities, catalyzing (i) the coupling of two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and (i) the conversion of prephytoene diphosphate into phytoene. We show now, with Capsicum chromoplast stroma, that the overall activity resides in a single protein, which has been purified to homogeneity by affinity chromatography. The monomeric structure and the molecular size (Mr 47,500) were demonstrated by NaDodSO4/PAGE and glycerol gradient centrifugation. Further characterization was achieved by using specific antibodies which allowed immunofractionation and immunoprecipitation of the enzymatic activity from chromoplast stroma. The two reactions followed conventional MichaelisMenten kinetics, with Km values of 0.30 ,.M and 0.27 IAM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence ofMn2. This selectivity may be one of the factors regulating the competition with potentially rival enzymes converting geranylgeranyl diphosphate into other plastid terpenoids. The two enzymatic reactions were inhibited by inorganic pyrophosphate and by the arginine-specific reagent hydroxyphenylglyoxal. In no instance were the two reactions kinetically uncoupled. These properties strongly suggest that the same enzyme catalyzes the two consecutive reactions, and we propose to name it phytoene synthase.Carotenoids are required for the normal differentiation and function of plastids. Phytoene, the first C40 intermediate in the carotenoid pathway, is synthesized by a soluble enzymatic complex localized in plastid stroma (1)(2)(3)(4)(5), by the multistep process outlined in Scheme I. The early steps, for which the individual enzymes were recently isolated and purified (6), are the isomerization of isopentenyl diphosphate (1) into dimethylallyl diphosphate (2), followed by three prenylations forming successively geranyl diphosphate (3), farnesyl diphosphate (4), and geranylgeranyl diphosphate (5). This sequence of reactions is common to all terpenoids, including other plastid terpenoids (chlorophylls, plastoquinones, tocopherols, phylloquinones, and polyterpenes). On the other hand, the next two steps, in which prephytoene diphosphate (6) and phytoene (7) are formed, are catalyzed by the first carotenoid-specific enzymes in the pathway, tentatively termed "geranylgeranyl pyrophosphate geranylgeranyltransferase" and "phytoene synthase" (7) since they have been neither isolated nor characterized previously from any other source (5,8,9). Therefore, the identification of the enzyme components catalyzing these two steps is crucial for our understanding of the molecular mechanism of carotenoid biosynthesis and of its regulation; a particularly interesting The present paper answers this question. We show, with Capsicum chromoplasts, previously selected for studies of carotenogenesis (3,(10)(11)(12), that a single monomeric protein ...

show abstract

Section: Resultssupporting

confidence: 61%

Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

Dogbo

Laferrière

d’Harlingue

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

show abstract

“…This work suggests therefore that positively charged intermediates, involved in the biosynthesis of a sterol, can be mimicked by ammonium (sulfonium, arsonium) groupcontaining molecules, resulting in a strong (e.g., chargecharge) interaction between the ammonium function and the active site of the enzyme. Such a conclusion is strengthened by a recent report showing that the farnesyl-pyrophosphate-squalene synthetase can be inhibited by a nitrogen-containing analogue of a carbocationic HEI involved in the reaction pathway (62). As shown in Figure 2, carbocationic species were frequently involved in the enzymology of sterol biosynthesis; the results here have important implications both for understanding the molecular mechanisms involved during the action of en-,zymes on sterol biosynthesis and for the search for new inhibitors.…”

Section: Resultssupporting

confidence: 57%

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Rahier¹,

Taton²,

Bouvier-Navé³

et al. 1986

Lipids

View full text Add to dashboard Cite

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ(8)-Δ(7)-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10(-8) M, Ki/Km=10(-3)) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.

show abstract

“…Modestly active inhibitors of squalene synthase have been described previously, and most are analogs of FPP (25)(26)(27)(28)(29) or are ammonium analogs of proposed carbocationic intermediates in the conversion of presqualene diphosphate to squalene (30,31). One potent exception to this was a famesyl phosphinylphosphonate ether analog (29), which was a competitive inhibitor of rat liver squalene synthase with a K1 of 37 nM.…”

Section: Discussionmentioning

confidence: 99%

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Bergstrom

Kurtz

Rew

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

315

158

View full text Add to dashboard Cite

Three closely related fungal metabolites, The mammalian isoprenoid pathway not only produces sterols but also produces dolichol, ubiquinone, the farnesyl group of heme A, the farnesyl and geranylgeranyl groups of prenylated proteins, and the isopentenyl side chain of isopentenyl adenine. The pathways for the synthesis of these other isoprenoids diverge from the synthesis ofcholesterol either at or before the farnesyl diphosphate (FPP) branch point. Thus, squalene synthase, which catalyzes the reductive dimerization of 2 mol of FPP to 1 mol of squalene (2, 3), is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase should serve to inhibit cholesterol synthesis and not adversely affect the synthesis of other isoprenoids. FPP, the substrate for squalene synthase, is water soluble and may be readily metabolized (4). Thus, squalene synthase offers a potential target for the safe and specific inhibition of cholesterol synthesis.In this report we describe the isolation, structure, physical characterization, and biological properties of three structurally similar fungal metabolites that are potent inhibitors of squalene synthase. These metabolites, zaragozic acid A (5-7), zaragozic acid B (8, 9), and zaragozic acid C (10-12), had been reported previously only in the patent literature; however, during the review process of this manuscript, three manuscripts (13-15) appeared on the squalestatins: squalestatin I is identical with zaragozic acid A, squalestatin II is des-4'-acetylzaragozic acid A, and squalestatin III is des-6-acylzaragozic acid A. This class of squalene synthase inhibitors has potential utility as cholesterol-lowering agents. MATERIALS AND METHODSZaragozic Acid A, Cultures, and Media. An unidentified sterile fungal culture, ATCC 20986, isolated from a water sample taken from the Jalon river in Zaragoza, Spain (hence the name zaragozic acids), was used to produce zaragozic acid A. The culture was maintained at 25°C on medium B agar slants composed of 4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, and 20 g of agar per liter at pH 7.0.Zaragozic acid A was produced in a two-tiered fermentation process consisting of mycelial growth and development in medium A of ref. 1 and product formation in medium C. Medium C contained 5 g of malt extract, 1 g of peptone, 15 g of dextrose, 1 g of KH2PO4, and 0.5 g of MgSO4 7H20 per liter. Fermentations consisted of mycelial growth in medium A for 72 hr at 250C with agitation, followed by inoculation (5-10%) of medium C. Maximum product was obtained from 14-day agitated fermentations at 250C.Isolation of Zaragozic Acid A. To isolate zaragozic acid A, 23 liters of harvested broth was filtered through Celite, and the mycelial cake was extracted twice with 7 liters of 50%o aqueous methanol. The filtrate was combined with the extracts, diluted with water to a final composition of 25% methanol, and adsorbed on a 1.5-liter column of Mitsubishi HP-20 resin. After a column wash with 6 liters of4:6 (vol/vol) methanol/water, crud...

show abstract

Squalene synthetase. Inhibition by an ammonium analogue of a carbocationic intermediate in the conversion of presqualene pyrophosphate to squalene

Cited by 48 publications

References 4 publications

Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Contact Info

Product

Resources

About