title: Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases.
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.
authors.affiliation: Department of Agronomy, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, KY 40546-0091, USA.
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Greenhagen
authors.forename: Paul
authors.initials: P
authors.lastname: Griggs
authors.forename: Shunji
authors.initials: S
authors.lastname: Takahashi
authors.forename: Lyle
authors.initials: L
authors.lastname: Ralston
authors.forename: Joe
authors.initials: J
authors.lastname: Chappell
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007535:Isomerases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D015203:Reproducibility of Results; D012717:Sesquiterpenes / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology
chemical_list: C471079:1-deoxycapsidiol; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C081843:capsidiol; C471077:germacrene A; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C436961:cytochrome P-450 CYP71D20 (Nicotiana tabacum); D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase; C108877:terpene synthase; D007535:Isomerases; C471078:premnaspirodiene synthase