Squalene 48-47 Isomerases

Fig. 5.1.2 Cholesterol biosynthesis branch of the isoprenoid biosynthetic pathway. Enzymes are numbered as follows 1 squalene synthase 2 squalene epoxidase 3 2,3-oxidosqua-lene sterol cyclase 4 sterol A24-reductase (desmosterolosis) 5 sterol C-14 demethylase 6 sterol A14-reductase (hydrops-ectopic calcification-moth-eaten, HEM, dysplasia) 7 sterol C-4 demethylase complex (including a 3/ -hydroxysteroid dehydrogenase defective in congenital hemidyspla-sia with ichthyosiform nevus and limb defects, CHILD, syndrome) 8 sterol A8-A7 isomerase (Conradi-Hunermann syndrome CDPX2) 9 sterol A5-desaturase (lathosterolosis) 10 sterol A7-reductase (Smith-Lemli-Opitz syndrome). Enzyme deficiencies are indicated by solid bars across the arrows...

One of the best therapeutic approaches may be to prevent absorption of cholesterol from the intestines by inclusion of a higher fiber content in the diet.66 Supplementation with a cholesterol-binding resin may provide additional protection. Plant sterols also interfere with cholesterol absorption. Incorporation of esters of sitostanol into margarine provides an easy method of administration. Supplemental vitamin E may also be of value.q Another effective approach is to decrease the rate of cholesterol synthesis by administration of drugs that inhibit the synthesis of cholesterol. Inhibitors of HMG-CoA reductase,s hh (e.g., vaLostatin) iso-pentenyl-PP isomerase, squalene synthase (e.g.,... 

Some isopentenyl pyrophosphate is converted to the isomer dimethylallyl pyrophosphate, by an isomerase that produces a mixture, isopentenyl pyrophosphate dimethylallyl pyrophosphate. From this point on, the carbon chain-length of the intermediates progressively increase this is followed by reduction to squalene, which has 30 carbon atoms in a folded chain and no oxygen atoms (Chap. 6). The conversion of isopentenyl pyrophosphate to squalene is shown in Fig. 13-22. 

Key to reactions (enzymes) 0, via mevalonate, isopentenyl phosphate, squalene 1, hydroxylase, desmolase (20,22) S, A -3j8-hydroxysteroid dehydrogenase, 5(4)-isomerase S, 21-hydroxylase 4, 17a-hydroxylase 6, ll 3-hydroxylase 6, desmolase (17,20) 7, 17 -hydroxysteroid dehydrogenase 8, 19-hydroxylase 9, aromatization. 

Mevalonate kinase Phosphomevalonate kinase Mevalonate PP decarboxylase GPP synthase IPP isomerase FPP synthase Squalene synthase... 

Steroids are members of a large class of lipid compounds called terpenes. Using acetate as a starting material, a variety of organisms produce terpenes by essentially the same biosynthetic scheme (Fig. 8). The self-condensation of two molecules of acetyl coenzyme A (CoA) forms acetoacetyl CoA. Condensation of acetoacetyl CoA with a third molecule of acetyl CoA, then followed by an NADPH-mediated reduction of the thioester moiety produces mevalonic acid [150-97-0] (72). Phosphorylation of (72) followed by concomitant decarboxylation and dehydration processes produce isopentenyl pyrophosphate. Isopentenyl pyrophosphate isomerase establishes an equilibrium between isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate (73). The head-to-tail addition of these isoprene units forms geranyl pyrophosphate. The addition of another isopentenyl pyrophosphate unit results in the sesquiterpene (C15) famesyl pyrophosphate (74). Both of these head-to-tail additions are catalyzed by prenyl transferase. Squalene synthetase catalyzes the head-to-head addition of two achiral molecules of famesyl pyrophosphate, through a chiral cyclopropane intermediate, to form the achiral triterpene, squalene (75). 

As part of the extensive studies by Cornforth and co-workers of squalene biosynthesis, the stereochemistry of the last step has now been elucidated. This concerns the stereochemistry of proton addition to isopentenyl pyrophosphate with formation of the new methyl group of dimethylallyl pyrophosphate under the influence of isopentenyl isomerase (EC 5.3.3.2). The investigation hinged on the generation of a chiral methyl. CHDT. [2- C,2R- H,3R]- and [2- C,2S- H,3/ ]-mevalonic acid were separately converted in the presence of... 

Prenyltransferase activities have been studied in C. roseus both at the enzyme level and at the product level. Biosynthetic capabilities were investigated by incubating [1- C]IPP with aliquots of cell-free homogenates prepared from P. aphanidermatum treated and untreated suspension-cultured cells of C. roseus. After elicitation, the total incorporation of IPP into prenyl lipids was decreased, in particular into squalene. But the incorporation of IPP into some (as yet unidentified) compounds was increased (99). The prenyltransferases and subsequent enzyme activities are relatively easily extracted and remain complexed so that the product of one enzyme can be used as a substrate for the next enzyme. With an assay for these enzymes as described in detail in Threlfall and Whitehead (101), about a dozen enzyme activities could be detected in series using cell-free preparations of elicited Tabemaemontana divaricata cells (27). In the elicited C. roseus cells, the activities of IPP isomerase, famesyl diphosphate synthase, squalene synthase, squalene-2,3-epoxidase (and probably also a squalene-2,3-epoxide cyclase) were thus detected. Compared with the control nontreated cells, squalene production seemed to be reduced particularly (99). 

For C. roseus suspension-cultured cells, elicitation with fungal elicitors results in the induction of TDC activity (99,186,202,203,284,329-331). This is due to the induction of expression of the Tdc gene. Similarly, SSS activity is induced (202,203,284,329,330). The induction by the Pythium aphanider-matum or yeast elicitor of the transcription of both genes is not affected by cycloheximide that is, the induction is independent of de novo protein biosynthesis, and thus follows an already available signal-transduction chain. The response is quite fast, for the enhanced transcription can already be measured 15 min after elicitation (202,203). Also, the NADPH cytochrome P-450 reductase mRNA level is induced by elicitation with fungal elicitors (113). Moreno et al (99,151) measured activities of a number of enzymes involved in secondary metabolism in C. roseus before and after elicitation with a P. aphanidermatum preparation. GlOH activity was found to be slightly decreased by elicitation and IPP-isomerase showed similar behavior. The pattern of terpenoids formed by the crude enzyme extracts from elicited and nonelicited cells was different. The total incorporation decreased, that is, the activities of the enzymes of the terpenoid pathway were lower. The relative incorporation decreased particularly for squalene. 

Target in sterol biosynthesis Sterol C14 demethylase A reductase and —> A isomerase 3-Keto reductase Squalene- epoxidase... 

In addition to the compound dependant inhibition profile, in different target pathogens different inhibition profiles are found that are characteristic for each individual species. Accordingly, the dominant site of inhibition after tridemorph application is the A A isomerase. With fenpropimorph, predominately the a reductase is targeted and A A isomerase is inhibited only at higher concentrations. With fenpropidin, predominately A reductase besides A A isomerase is inhibited [100]. At high concentrations the accumulation of squalene and 2,3-oxidosqualene indicates also the inhibition at earlier steps in sterol biosynthesis. 

In addition to the squalene oxide to cycloartenol cyclase, the set of phytosterol enzymes- the S-adenosyl-L-methionine C-24 methyl transferases (SMT) and the 9,19-cyclopropyl to 8- isomerase (COI) are considered as phylogenetically significant to plant evolution. The plant SMT is assumed to catalyze Re-face methylation of bent... 

Isopentenyl pyrophosphate is potentially a bifunctional molecule. Its terminal vinyl group gives a nucleophilic character whereas when it isomerizes to 3,3-dimethylallyl diphosphate, the latter is electrophilic. Thus, longer-chain polyprenyls are formed by a favourable condensation of isopentenyl pyrophosphate first with dimethylallyl pyrophosphate and later with other allylic diphosphates. The initial interconversion of isopentenyl diphosphate and dimethylallyl diphosphate is promoted by an isomerase. The successive condensations yield the Cio compound geranyl diphosphate and then the C15 farnesyl diphosphate. The two molecules of famesyl diphosphate condense to form presqualene pyrophosphate which is reduced by NADPH to give the C30 open chain terpenoid squalene. The condensation reactions with IPP are a rather novel method of C-C bond formation since in the formation of other types of natural products (peptides, sugars, fatty acids, etc.) the reactions involve Claisen- or aldol-type condensations. 

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