| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
During the research period (2001-2002), we got some significant achievements for squalene hopene cyclase as follows.
(1) Site-directed mutagenesis experiments targeted for Tyr495, Tyr612 and Tyr609 showed that the role of these amino acids was ascribed to the reinforcement of the role of Asp377 and Phe365 which works for the cation-π interaction.
(2) Alteration of the bulk size at 420 and 607 into the larger amino acids allowed the creation of unnatural natural product, named neoachillapentaene, which was produced by the boat structure during the cyclization process. This finding indicates that the steric bulk size of crucial amino acids perturbs the substrate folding.
(3) We synthesized a 2,3-oxidosqualene analogue which have ethyl group at 10-position. This analog was cyclized by lanosterol synthase to give the abnormal cyclization products with trimethylcyclohexane moiety, which produced through the boat-folding structure and produced by the catalysis of 3R-oxidosqualene. Lanosterol synthase is rigorously specific to 3S-oxidosqualene and inert to the 3R-form. This investigation gave a deeper insight into the substrate recognition by lanosterol synthase.
(4) We also synthesized C(10) norsqualene, which was subjected to the enzymic reaction by squalene-hopene cyclase. This enzymic reaction afforded novel carbocyclic skeleton(s), i. e. 6/5+5/5+(6). This finding also indicated that the bulk size of the substrate also significantly influence the cyclization pathway.
(5) Comparison of amino acid alignment between squalene and lanosterol cyclases encouraged to delete Gly600 of the squalene cyclase. This deleted mutant was created by the PCR method. Squalene cyclase catalyzes 3 substrates, i. e. squalene (original substrate), 3S- and 3R-oxidosquaene. However, this mutant was only active to 3S-oxidosquaene. This behavior was analogous to lanosterol synthase. Thus, we have succeed in altering the substrate specificity.
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