2.3- oxidosqualene:lanosterol cyclase

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. 

Squalene synthase, 3 = Squalene monooxygenase, 4 = 2,3-Oxidosqualene lanosterol cyclase, 5 = Enzymes catalyzing 20 separate reactions. Note that squalene and lanosterol are acted upon by ER membrane enzymes while they are bound to carrier proteins in the cytoplasm. 

Figure 14.5-1. Synthesis of lanosterol analogues using 2,3-oxidosqualene lanosterol cyclase.

In plants, sterols arise through the intermediacy of cycloartenol (3) and 2,3-oxidosqualene cycloartenol cyclase (EC 5.4.99.8), whereas, in animals, sterols arise from lanost-erol (15) and 2,3-oxidosqualene lanosterol cyclase (EC 5.4.99.7) (Figs. 23.7 and 23.8). Despite several reports (all... 

All steroids are biosynthesized from the triterpene squalene, which is converted to an epoxide by the enzyme squalene 2,3-epoxidase. In mammals, this is converted to lanosterol by 2,3-oxidosqualene-lanosterol cyclase. You should find the first step of the process fairly easy to follow, as it is simply a cascade of electrophilic additions to double bonds. The next step is a cascade of rearrangements— come back and look at that again, once you have studied rearrangements in Chapter 18, as at this point, it may seem a bit mysterious (Figure 16.9). There are 7 stereocenters in lanosterol, which gives a potential for 128 stereoisomers, but a single stereoisomer is produced by the enzymatic reaction. 

Irrespective of whether the oxidative cyclization of pCA is a concerted reaction or involves two (or more) steps, the reaction requires a single enzyme molecule. This conversion, therefore, differs from squalene oxidative cyclization, which requires an enzyme for the oxidative step and another enzyme (2,3-oxidosqualene lanosterol cyclase) for the cyclization (Singer et aL, 1956). The possibility that the oxidative cyclization of pCA may be a true concerted (one step) process cannot be ruled out at this stage. It may explain the specificity (Steenkamp et aL, 1974) of the enzyme for pCA as substrate and the observation that in all reaction mixtures aCA was formed at approximately the same rate as the rate of dehydrogenation of the electron acceptor. 

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. 

Fig. 14.12. Enzymatic transformation of acyclic squalene oxide (A) into tetracyclic lano-sterol (G). The oxidosqualene-lanosterol cyclase controls the conformation of the substrate so effectively that only one out of 64 possible diastereomers is formed.

Squalene is converted into the first sterol, lanosterol, by the action of squalene epoxidase and oxidosqualene lanosterol cyclase. The catalytic mechanism for the cyclase s four cyclization reactions was revealed when the crystal stmcture of the human enzyme was obtained (R. Thoma, 2004). Oxidosqualene lanosterol cyclase is considered an attractive target for developing inhibitors of the cholesterol biosynthetic pathway because its inhibition leads to the production of 24,25-epoxycholesterol (M.W. Huff, 2005). This oxysterol is a potent ligand activator of the liver X receptor (LXR) and leads to expression of several genes that promote cellular cholesterol efflux, such as ABCAl, ABCG5, and ABCG8 (Section 4.1). Thus, inhibitors of oxidosqualene lanosterol cyclase could be therapeutically advantageous because they would reduce cholesterol synthesis and promote cholesterol efflux (M.W. Huff, 2005). 

Abe 1, Prestwich GD (1999) Squalene epoxidase and oxidosqualene lanosterol cyclase-key enzymes in cholesterol biosynthesis. In Cane DE (ed) Comprehensive natural products chemistry isoprenoids including carotenoids and steroids. Elsevier Science, Oxford, Vol 2, p 267... 

Purification. The first development is the purification, to apparent homogeneity, of several cyclase enzymes from natural sources. Abe, Ebizuka and coworkers worked out methods for the purification of oxidosqualene-p-amyrin and oxidosqualene-cycloartenol cyclases from Piswn sativum and Rabdosia Japonica, and of oxidosqualene-lanosterol cyclase from rat liver (31-34). These purification... 

Comparison of Squalene-Hopene and Oxidosqualene-Lanosterol Cyclase Sequences. 

The predicted amino acid sequences for the squalene-hopene and oxidosqualene-lanosterol cyclases are presented in Figure 4. A comparison of these sequences has important implications for the evolution and mode of action of these enzymes. The squalene-hopene cyclase is predicted to consist of 627 amino acids with a molecular... 

Figure 4. Predicted amino acid sequence for the squalene-hopene cyclase from B. acidocaldarius (Top) and the oxidosqualene-lanosterol cyclase from C. albicans (Bottom). Regions of sequence identity are underlined. Hydrophobic regions of Ae C. albicans cyclase are italicized. Tryptophan residues (W) are bold.

Figure 5. Hydropathy plots for the squalene-hopene (top) and oxidosqualene-lanosterol (bottom) cyclases. Arrows indicate notably hydrophobic regions of the oxidosqualene-lanosterol cyclase primary sequence.

Amino Acid Sequence Identity. The predicted amino acid sequences for the squalene-hopene and oxidosqualene-lanosterol cyclases have no significant similarities to sequences in protein sequence databases PIR 31 and SWISS-PROT 21 (40,42). However, when the predicted amino acid sequences of the cyclases are compared to one another, four regions of substantial similarity are observed. As... 

Figure 6. Regions of predicted amino acid sequence identity between squalene-hopene and oxidosqualene-lanosterol cyclase enzymes.

The Aromatic Hypothesis. In addition to the specifically conserved tryptophan residues, we had previously noted that the squalene-hopene cyclase and the oxidosqualene-lanosterol cyclase are generally rich in both tprptophan and tyrosine, amino acid residues with electron-rich aromatic sidechains (42), The squ ene-hopene cyclase contains 3.2% tryptophan and 4.0% tyrosine. The median values for representation of these amino acids in E, coli proteins are 1.2% and 2.7%, respectively (43). The levels of tryptophan and tyrosine found in the B. acidocaldarius enzyme exceed those found in > 95% and 85% of E, coli proteins, respectively. The oxidosqualene-lanosterol cyclase contains 3.0% ti tophan and 6.3% tyrosine. When compared to proteins from the yeast S. cerevisiae (for which the me an values for representation of tryptophan and tyrosine are 0.9% and 3.3%, respectively), the C. albicans enzyme i ssesses these amino acids at levels greater than those found in 99% of all S. cerevisiae proteins. 

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. 

In contrast to the above results the substrates (23) and (25) were transformed enzymically by 2,3-oxidosqualene sterol cyclase to the corresponding lanosterol derivatives (27) and (28). In addition, 6-demethyl-2,3-oxidosqualene underwent enzymic cyclization to 19-norlanosterol (29). Van Tamelen and Freed ... 

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... 

---