There has been remarkable advances in the studies of oxidosqualene and squalene cyclases in the past ten years. First, complete purifications of the enzymes have been attained from several species such as vertebrates, plant, yeast and bacterium, irrespective of unstable and membrane-bound nature. Second, application of cDNA cloning technique to the cyclases have succeeded in determining the alignments of amino acids from various biological sources including human. Third, the substrate analogues including the suicide inhibitors have made a great contribution to the polycyclization mechanism. In this symposium, we report a definitive evidence that the polycyclization proceeds via the discrete carbocationic intermediate formed during the cyclization, but not via a concerted manner as proposed before; substitution of methyls with ethyl groups at 10- and 15-positions 6 halted the enzymic reaction at the monocyclic stage 7 and 8. Substrate analogue 9 with ethyl group at 15-position afforded 10 (normal cyclization) and 11 (tricyclic 6/6/5), the latter being formed under the control of Markovnikov rule. This is in contrast to the protosterol cation, which is formed under anti-Markovnikov control. Thus, oxidosqualene would be converted via the 5-membered intermediate 12 under the control of Markovnikov rule, which then undergoes a ring expansion to form 6-membered C-ring. Mechanisms of the formation of tetrahymanol and hopene from squalene itself are discussed, especially on the terminal cyclization (E-ring formation). Substrate analogs 14, 15, 18, 22 have demonstrated the cyclization mechanisms as follows: for tetrahymanol cyclase, the terminal cyclization proceeds by the process of stereoelectronic control, suggesting little participation of the enzyme, while hopene cyclase strongly binds to the terminal methyl groups to form 5-membered E-ring. From our studies, methyl groups of the substrate seem to bind the cyclase enzymes and the substrates were then subjected to the folding of chair-boat or chair-chair inside the enzymes leading to production of the desired triterpene skeletons. Over-expression of hopene cyclase was achieved successfully and the point mutations of amino acids in QW motif proved that the functions of D and W are crucial for the enzyme activity.
