Biochemical conservation and evolution of germacrene A oxidase in asteraceae.
Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.
Nguyen
Don Trinh
DT
Department of Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada.
Göpfert
Jens Christian
JC
Ikezawa
Nobuhiro
N
Macnevin
Gillian
G
Kathiresan
Meena
M
Conrad
Jürgen
J
Spring
Otmar
O
Ro
Dae-Kyun
DK
D019659:Asteraceae / Q000201:enzymology*; D002384:Catalysis; D002855:Chromatography, Thin Layer / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D014644:Genetic Variation; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry* / Q000235:genetics
D010940:Plant Proteins; D011994:Recombinant Proteins; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases