On the cyclization mechanism of squalene: a ring expansion process of the five-membered D-ring intermediate

Abstract

Site-directed mutagenesis experiments with W169F, W169H and W489F for the squalene-hopene cyclase, and the formation of 10 possessing the five-membered D-ring and a tetrahydrofuran moiety as the enzyme product of the analogue 8 with a hydroxy group, strongly suggest that a ring expansion reaction from the five- to the six-membered ring is responsible for the D-ring formation of hopene.

References

1. I. Abe, M. Rohmer and G. D. Prestwich, Chem. Rev., 1993, 93, 2189.
2. G. Ourisson, M. Rohmer and K. Poralla, Annu. Rev. Microbiol., 1987, 41, 301.
3. C. Feil, R. Sussmuth, G. Jung and K. Poralla, Eur. J. Biochem., 1996, 242, 51.
4. K. U. Wendt, K. Poralla and G. E. Schulz, Science, 1997, 277, 1811.
5. T. Sato, Y. Kanai and T. Hoshino, Biosci. Biotechnol. Biochem., 1998, 62, 407.
6. All the HRMS (EI) and NMR (H-H COSY 45, HOHAHA, NOESY, DEPT, HMQC and HMBC) spectra were consistent with the proposed structures of 4 and 10.
7. E. J. Corey and H. Cheng, Tetrahedron Lett., 1996, 37, 2709; E. J. Corey, S. C. Virgil, H. Cheng, C. H. Baker, S. P. T. Matsuda, V. Singh and S. Sarshar, J. Am. Chem. Soc., 1995, 117, 11 819; T. Hoshino and Y. Sakai, Chem. Commun., 1998, 1591.
8. I. Abe and M. Rohmer, J. Chem. Soc., Perkin Trans. 1, 1994, 783.
9. I. Abe, T. Dang, Y. F. Zheng, B. A. Madden, C. Fei, K. Poralla and G. D. Prestwich, J. Am. Chem. Soc., 1997, 119, 11 333.
10. K. Poralla, Bioorg. Med. Chem. Lett., 1994, 4, 285; I. Abe and G. D. Prestwich, Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 9274; D. A. Dougherty, Science, 1996, 271, 163.
11. The mutations gave a lowering of the optimal temperature, but no change with pH. Reactions with 5 µg of purified SHC were conducted at 30 °C and pH 6.0 for 1 h; thermal denaturation of the SHCs was not found. The kinetic values of K_m and V_max were determined from Lineweaver–Burk plots as follows: K_ms: 16.7, 277, 280 and 92 µM; and V_maxs: 0.09, 0.078, 0.045 and 0.017 nmol min^–1µg^–1, respectively, for the wild-type, W169F, W169H and W489F.