TAILIEUCHUNG - Báo cáo khoa học: Identification of b-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the ini-tial origin of structural diversity of skeletons in their biosynthesis, and sub-sequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. | iFEBS Journal Identification of b-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay Masaaki Shibuya1 Masaki Hoshino1 Yuji Katsube1 Hiroaki Hayashi2 Tetsuo Kushiro1 and Yutaka Ebizuka1 1 Graduate Schoolof PharmaceuticalSciences The University of Tokyo Japan 2 Gifu PharmaceuticalUniversity Japan Keywords b-amyrin 24-hydroxylase CYP93E1 Glycine max P450 sophoradiol24-hydroxylase Correspondence Y. Ebizuka Graduate Schoolof PharmaceuticalSciences The University of Tokyo Hongo Bunkyo-ku Tokyo 113-0033 Japan Fax 81 3 5841 4744 Tel 81 3 5841 4740 E-mail yebiz@ Received 28 October 2005 revised 12 December 2005 accepted 23 December 2005 doi Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes five genes CYP71D8 CYP82A2 CYP82A3 CYP82A4 and CYP93E1 reported to be elicitor-inducible genes in Glycine max expressed sequence tags EST were amplified by PCR and screened for their ability to hydroxylate triterpenes b-amyrin or sophora-diol by heterologous expression in the yeast Saccharomyces cerevisiae. Among them CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as and soyasapogenol B respectively by GC-MS. Co-expression of CYP93E1 and b-amyrin synthase in S. cerevisiae yielded olean-12-ene-3b 24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a .

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