Lecture by Dae-Kyun Ro: "Chemical Diversity and Evolution Sunflower Family" : 31. maj 2011
Lecture by visiting speaker Dae-Kyun Ro, Assistant Professor, Department of Biological Sciences, University of Calgary.
The title of the talk: "Chemical Diversity and Evolution Sunflower Family"
When: Tuesday 31st May, 13:00-14:00
Abstract: Asteraceae (or Compositae) is the largest plant family which constitutes 8% (~23,000 species) of flowering plants on earth. It first emerged from South America at about 50 million years ago and has become dominant cosmopolitan plants as represented by sunflower and dandelion. One characteristic chemical constituent of Asteraceae is sesquiterpene lactone (STL). Thousands of bioactive STLs derived from several C-15 core structures have been documented, but their biochemical mechanism is poorly understood. We postulated that studying STLs in Asteraceae could provide an insight into the adaptive evolution of enzymes that has led to the enormous chemical diversification in this family. Natural variants of closely related enzymes, such as cytochrome P450 monooxygenases (P450s), will be excellent templates for the evolution-inspired structure-function analysis. In order to better understand the catalytic evolution in the STL biosynthesis, we first focused on the conservation of the key P450 enzyme that catalyzes three-step oxidations of the C12 carbon of sesquiterpene backbone, hence adding a C12 carboxylic acid moiety. Homologs of Artemisia annua amorphadiene C12 oxidase were cloned from three major subfamilies (Asteroideae, Cichorioideae, and Carduoideae) and also from a basal lineage (Barnadesioideae) of Asteraceae. These were functionally expressed in yeast synthesizing the substrate, germacrene A. All of the recombinant P450s were able to catalyze the conversion of germacrene A to germacrene A acid (GAA), substantiating that this three-step oxidation activity is conserved at the basal lineage of Asteraceae. Interestingly, these germacrene A oxidases (i.e., evolutionary predecessors of amorphadiene oxidase) showed promiscuous activities toward various non-natural substrate, but the later-evolved amorphadiene oxidase showed activity only for amorphadiene. Using this new P450, the yeast strain was further engineered to produce GAA to look for the lactone-forming enzymes. For this purpose, transcripts from sunflower (Helianthus annuus) trichome were sequenced, and a P450 clone encoding 8-beta-GAA hydroxylase was functionally identified. Its closest homolog in lettuce (Lactuca sativa) was also identified; however, this lettuce P450 catalyzed the 6-alpha-hydroxylation of GAA, which result in non-enzymatic formation of the simplest STL, costunolide. Homology modeling of these two P450s suggests that subtle residue variations in the active site appear provide regio- and stereo-selectivity of these two related enzymes. Evolutionary significance of these 8-beta- and 6-alpha-GAA hydroxylases will be discussed in the context of Asteraceae phylogeny.