2,3 -oxidosqualene

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. 

Lanosterol biosynthesis begins with the selective conversion of squalene to its epoxide, (35)-2,3-oxidosqualene/ catalyzed by squalene epoxidase. Molecular 02 provides the source of the epoxide oxygen atom, and NADPH is required, along with a flavin coenzyme. The proposed mechanism involves... 

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. 

To summarize, squalene epoxidase is a flavoprotein capable of catalyzing the insertion of oxygen into the 2,3-double bond of squalene to give 2,3-oxidosqualene, with the second oxygen atom from 02 being reduced to water. The reducing equivalents necessary for this transformation are relayed from NADPH through NADPH-cytochrome c reductase to the flavin cofactor of the epoxidase. 

Both compounds discussed in this section, oxidosqualene and epoxystyrene, are intermediates in metabolic pathways. That is to say that the epoxide is not found in the final product but rather serves to impart a specific reactivity to the molecule that is vital for the subsequent reaction step. The epoxide in oxidosqualene can be viewed as a masked cation that is required in order to initiate a series of C-C bond-... 

Despite the broad medical potentials reported so far, the total synthesis of triterpene QMs is yet to be reported. On the contrary, the biosynthesis of triterpene QMs has recently been validated as from the oxidosqualene 88 (Scheme 8.16) in the plants including Maytenus aquifolium and Salacia campestris.10S With the assistance of HPLC analysis and isotopic labeling, it was found that triterpene QMs 90 were formed only in the root of these plants from friedelin 89 and similar cyclized intermediates, which were synthesized in the leaves from oxidosqualene by cyclase. 

An example of this type is the highly stereoselective formation of lanosterol (0-6) from (S)-2,3-oxidosqualene (0-5) in Nature, which seems not to follow a concerted mechanism (Scheme 0.2) [9],... 

The sequence of transformations from squalene to lanosterol begins by the enzymatic oxidation of the 2,3-double bond of squalene to form (3S)-2,3-oxidosqualene [also called squalene 2,3-epoxide]. 

CYCLIZATION OF 2,3-OXIDOSQUALENE - THE FIRST COMMITTED STEP IN TRITERPENOID BIOSYNTHESIS... 

Triterpenoid saponins are synthesized via the isoprenoid pathway.4 The first committed step in triterpenoid saponin biosynthesis involves the cyclization of 2,3-oxidosqualene to one of a number of different potential products (Fig. 5.1).4,8 Most plant triterpenoid saponins are derived from oleanane or dammarane skeletons although lupanes are also common 4 This cyclization event forms a branchpoint with the sterol biosynthetic pathway in which 2,3-oxidosqualene is cyclized to cycloartenol in plants, or to lanosterol in animals and fungi. 

Fig. 5.1 Cyclization of 2,3-oxidosqualene to sterols and triterpenoids. The 2,3-oxidosqualene cyclase enzymes that catalyse the formation of the different products are indicated LS, lanosterol synthase CS, cycloartenol synthase LuS, lupeol synthase PAS, P-amyrin synthase aAS, a-amyrin synthase.

Characterization of fi-Amyrin Synthase from Avena strigosa-A Novel Oxidosqualene Cyclase... 

Genetic analysis indicates that two of the 10 sad mutants of A. strigosa that we isolated represent different mutant alleles at the Sadi locus.6 These mutants accumulate radiolabelled 2,3-oxidosqualene but not p-amyrin when the roots are fed with 14C-labelled precursor mevalonic acid, suggesting that the triterpenoid pathway is blocked between 2,3-oxidosqualene and P-amyrin.34 The roots of these mutants also lack detectable P-amyrin synthase activity, but, like the wild type and the other mutants, are unimpaired in cycloartenol synthase (CS) activity and sterol biosynthesis.34 The transcript levels for AsbASl are substantially reduced in roots of sadl mutants, while AsCSl transcript levels are unaffected,35 suggesting that the sadl mutants are either mutated in the AsbASl gene itself or in a gene involved in its regulation. 

KUSHIRO, T., SHIBUYA, M., EBIZUKA, Y P-Amyrin synthase. Cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants, Eur. J. Biochem., 1998,256,238-244. 

HUSSELSTEIN-MULLER, T., SCHALLER, H., BENVENISTE, P., Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana, Plant Mol. Biol., 2001, 45, 75-92. 

SEGURA, M.J.R., MEYER, M.M., MATSUDA, S.P.T., Arabidopsis thaliana LUP1 converts oxidosqualene to multiple triterpene alcohols and a triterpene diol, Org. Letts., 2000, 2, 2257-2259. 

MATSUDA, S.P.T., On the diversity of oxidosqualene cyclases. In Biochemical Principles and Mechanisms of Biosynthesis and Degradation of Polymers (A. Steinbuchel, ed,), Wiley-VCH, Weinheim. 1998, pp. 300-307. 

HENRY, M., RAHIER, A., TATON, M., Effect of gypsogenin 3,0-glucuronide pretreatment of Gypsophila paniculata and Saponaria officinalis cell suspension cultures on the activities of microsomal 2,3-oxidosqualene cycloartenol and amyrin cyclases, Phytochemistry, 1992,31,3855-3859. 

TATON, M., BENVENISTE, P., RAHIER, A., N-[(l,5,9)-tnmethyl-decyl]-4a,10-dimethyl-8-aza-trans-decal-3P-ol A novel potent inhibitor of 2,3-oxidosqualene cycloartenol and lanosterol cyclases, Biochem. Biophys. Res. Comm., 1986, 138, 764-770. 

CATTEL, L., CERUTI, M., 2,3-Oxidosqualene cyclase and squalene epoxidase Enzymology, mechanism and inhibitors. In Physiology and Biochemistry of Sterols (G.W. Patterson and W.D. Nes, eds,), American Oil Chemists Society, Champaign. 1992, pp. 50-82. 

HARALAMPIDIS, K, BRYAN, G Qi, X., PAPADOPOULOU, K., BAKHT, S, MELTON, R, OSBOURN, A.E., A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots, Proc. Natl. Acad. Sci., USA, 2001,98,13431-13436. 

PORALLA, K HEWELT, A., PRESTWICH, G.D., ABE, I., REIPEN, I., SPRENGER, G., A specific amino acid repeat in squalene and oxidosqualene cyclases, TIBS, 1994, 19,157-158. 

FANG, T-Y., BAISTED, D.J., 2,3-Oxidosqualene cyclase and cycloartenol-S-adenosylmethionine methyltransferase activitites in vivo in the cotyledon and axis tissues of germinating pea seeds, Biochem. J., 1975,150, 323-328. 

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