Squalene epoxide cyclases

Figure 21.29 shows the conversion of squalene to cholesterol. The details of this conversion are far from simple. Squalene is converted to squalene epoxide in a reaction that requires both NADPH and molecular oxygen (O2). This reaction is catalyzed by squalene monooxygenase. Squalene epoxide then undergoes a complex cyclization reaction to form lanosterol. This remarkable reaction is catalyzed by squalene epoxide cyclase. The mechanism of the reaction is a concerted reaction—that is, one in which each part is essential for any other part to take place. No portion of a concerted reaction can be left out or changed because it all takes place simultaneously rather than in a sequence of steps. The conversion of lanosterol to cholesterol is a complex process. It is known that 20 steps are required to remove three methyl groups and to move a double bond, but we shall not discuss the details of the process. 

B Formation of Tetracyclic and Pentacyclic Triterpenes from Squalene Epoxide. In most microorganisms and in plants and animals the cyclic triterpenes are derived from squalene-2,3-epoxide, which is formed from squalene by a monooxygenase (C 2.6.5). The action of different, squalene epoxide cyclases results in the formation of different types of cyclic triterpenes. 

Figure 1.7 illustrates the synthesis of sterols in yeasts. Basically, sterols are synthesised by the mevalonate pathway. The key stage in this pathway is, without any doubt, the reaction catalysed by squalene monooxygenase. This reaction, which uses oxygen as substrate, transforms squalene into squalene 2,3, epoxide. Later, squalene epoxide lanosterol cyclase catalyses the synthesis of the first sterol of the pathway. 

A comparison of the efficiency of the 2,3-oxidosqualene cyclase from human placenta microsomes with that from rat liver microsomes has led to the conclusion that in human placenta the conversion of squalene into lanosterol is limited by the rate of squalene epoxidation. Tracer from [l- C]-2,3-oxidosqualene was incorporated into cycloartenol (1% yield) by a cell-free system from Alnus glutinosa but none of the triterpenoid glutinone was synthesized from the labelled precursor. When 2,3-oxidosqualene was incubated with cell-free extracts from corn embryos the only product was cycloartenol, whereas when l-trarts-T-nor-2,3-oxidosqualene (9) was the substrate both 31-norcycloartenol and 31-norlanosterol were formed " l-c/s-T-nor-2,3-oxidosqualene (10) gave rise to no detectable cyclization products... 

The final stage of cholesterol biosynthesis starts with the cyclization of squalene (Figure 26.11). Squalene is first activated by conversion into squalene epoxide (2,3-oxi-dosqualene) in a reaction that uses O2 and NADPH. Squalene epoxide is then cyclized to lanosterol by oxi-dosqualene cyclase. This remarkable transformation proceeds in a concerted fashion. The enzyme holds squalene epoxide in an appropriate conformation and initiates the reaction by protonating the epoxide oxygen. The carbocation formed spontaneously rearranges to produce lanosterol. Lanosterol is converted into cholesterol in a... 

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). 

Abe, I., Y. Ebizuka, S. Seo, and U. Sankawa, Purification of squalene-2,3-epoxide cyclases from cell suspension cultures of Rabdosia japonica Kara, FEES Lett., 249, 100-104 (1989b). 

Formation of steroids of the lanosterol-cycloartenol-cucurbitacin-type probably starts with squalene epoxide in a chair-boat-chair-boat-unfolded conformation (Fig. 115). It depends on the location of the proton that is split off, whether the cyclase forms lanosterol (animals, microorganisms), cycloartenol (plants), or cucurbitacins (Cucurbitaceae, Cruciferae), e.g., cucurbitacin E ( x-elaterin). 

Squalene monooxygenase, an enzyme bound to the endoplasmic reticulum, converts squalene to squalene-2,3-epoxide (Figure 25.35). This reaction employs FAD and NADPH as coenzymes and requires Og as well as a cytosolic protein called soluble protein activator. A second ER membrane enzyme, 2,3-oxidosqualene lanosterol cyclase, catalyzes the second reaction, which involves a succession of 1,2 shifts of hydride ions and methyl groups. 

The second part of lanosterol biosynthesis is catalyzed by oxidosqualene lanosterol cyclase and occurs as shown in Figure 27.14. Squalene is folded by the enzyme into a conformation that aligns the various double bonds for undergoing a cascade of successive intramolecular electrophilic additions, followed by a series of hydride and methyl migrations. Except for the initial epoxide protonation/cyclization, the process is probably stepwise and appears to involve discrete carbocation intermediates that are stabilized by electrostatic interactions with electron-rich aromatic amino acids in the enzyme. 

Head-to-head condensation of two farnesylpyrophosphate (C]3—PP) molecules yields a G13-cyclopropane (C3)-CH intermediate which is then reduced to yield squalene H(CH2-C(CH3)=CH-CH2)3-(CH2-CH=C(CH3)CH2)3 (C30), that is, if one represents the isoprene polarities as IP and PI, one could represent squalene as (IP)3—(PI)3. Squalene is subsequently oxidized [via a squalene monooxygenase] to yield squalene 2,3-epoxide which is cyclized to the tetracyclic sterol terpene lanosterol (C30) [via squalene cyclase]. 

Cyclisation of squalene 2,3-epoxide (10) yielded, in addition to tricyclic products, the bicyclic alcohol (11) which presumably arises from the bicyclic carbonium ion (12) by a series of hydride and methyl migrations. Sharpless and van Tamelen have suggested that one of the functions of the cyclase enzyme is to prevent this type of process at the bicyclic level. 

Squalene 2,3-epoxide has been isolated from the green alga Caulerpa prolifera. Oxidation of squalene with t-butyl hydroperoxide in the presence of Mo02(acac)2 and di-isopropyl (+)-tartrate gave the 2,3-epoxide (31%) with an induced asymmetry of about 14% in favour of the (35)-isomer. The ability of oxidosqualene cyclases to accept unnatural precursors has been further extended by the observation that lanosterol cyclase from rabbit liver converts the synthetic epoxide (1) into the jS-onocerin derivative (2). An authentic sample of (2) was prepared by sodium cyanoborohydride reduction of /3-onoceradione... 

Cycloartenol (3) is the major product of condensation of squalene 2,3-epoxide (2) by a chair-boat-chair-boat transition state in plants. This condensation is effected by squa-lene-2,3-oxide-cycloartenol cyclase (Fig. 23.6). Micro-somes from Rabdosia japonica (Acanthaceae) showed activity for cyclizing squalene 2,3-epoxide into cycloartenol, 3-amyrin, and a-amyrin. Purified cycloartenol cyclase had a molecular weight of 54,000 (Abe et al., 1989a, 1989b). 

Squalene monooxygenase transforms the double bond on one end of the squalene into an epoxide. Then the cyclase enzyme catalyzes the closing of the rings. 

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