Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases

Bryan T. Greenhagen, Paul Griggs, Shunji Takahashi, Lyle Ralston, Joe Chappell

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)385-394
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume409
Issue number2
DOIs
StatePublished - Jan 15 2003

Bibliographical note

Funding Information:
The Kentucky Agricultural Experiment Station and a grant from the NSF supported this work.

Keywords

  • 5-epi-Aristolochene dihydroxylase hydroxylase
  • 5-epi-Aristolochene synthase
  • Capsidiol
  • Germacrene A
  • Germacrene diol
  • Premnaspirodiene synthase
  • Solavetivone

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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