Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases
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- 作者：Greenhagen ; Bryan T. ; Griggs ; Paul ; Takahashi ; Shunji ; Ralston ; Lyle ; Chappell ; Joe
- 关键词：5-epi-Aristolochene synthase ; Premnaspirodiene synthase ; 5-epi-Aristolochene dihydroxylase hydroxylase ; Germacrene A ; Capsidiol ; Germacrene diol ; Solavetivone
- 刊名：Archives of Biochemistry and Biophysics
- 出版年：2003
- 出版时间：January 15, 2003
- 年：2003
- 卷：409
- 期：2
- 页码：385-394
- 全文大小：291 K
文摘
5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.