Functional analyses of terpene-related biosynthetic enzymes and creaion of novel cyclases

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This project was to elucidate the function of squalene-hopene cyclase and terpene-related biosynthetic enzymes and to create a novel terpene cyclase.1.SHC (squalene-hopene cyclase)(1)final deprotonation occurs from the terminal (Z)-Me and highly polarized water acted to abstract the proton.(2)Squalene analogs having a methyl group at different positions were synthesized in order to know how the methyl group on squalene affects the polycyclization. Introduction of Me group at 7- or 11-position resulted in no reaction.(3)SHC can accept truncated analogs having C15,C20 and C25, giving sesqui, di- and sesterterpenes.(4)We first succeeded in alteration of substrate specificity. Deletion of Gly600 alters the substrate specificity into eukaryotic type cyclase specific to (3S)-2,3-oxidosqualene.(5)Protocol of the site-specific incorporation of unnatural amino acid was developed by us. Incorporation of fluorophenylalanines at positions 605 and 365 established that cation-π interaction play a critical role for the cyclization cascade.2.Diterpene synthase from Mycobacterium tuberculosis(1)Rv3377c was cloned in E.coli. With aid of chaperon, this gene product was functionally expressed and determined to be diterpene cyclase after incubating various substrates. This product has halimane skeleton and was named tuberculosinol.(2)It turned out that Rv3378c was responsible for dephosphorylating tuberculosinol-pp.