The evolution of mono- and sesquiterpene diversity in maize
JÖRG DEGENHARDT
Martin-Luther-Universität Halle-Wittenberg
Institut für Pharmazie
Abteilung Molekulare Biotechnologie
Wolfgang-Langenbeck-Str. 4
06120 Halle
joerg.degenhardt@pharmazie.uni-halle.de
http://www.pharmazie.uni-halle.de/institutsbereiche/
JONATHAN GERSHENZON
Max-Planck-Institut
für Chemische Ökologie
Beutenberg-Campus
Hans-Knöll-Straße 8
D-07745 Jena
gershenzon@ice.mpg.de
http://www.ice.mpg.de/ger/home/home_en.htm
Terpenes are the largest group of plant secondary compounds, comprising about 30,000 structures that play numerous roles in primary metabolism and ecological interactions. Their large structural and functional diversity make them an excellent model to study chemical diversity in plants. The key step of terpene biosynthesis is catalyzed by the enzyme class of terpene synthases. Terpene synthases employ an electrophilic reaction mechanism that is able to form the large diversity of terpene products. In all plant species studied so far, terpene synthases are encoded by a large family of genes that share a common evolutionary origin and evolved to form complex terpene blends. Thus, this gene family is valuable tool to study the structural and functional diversification of these genes that results in the formation of a large variety of products.
To study the evolution of terpene diversity, we characterized the terpene synthases gene family in maize. After biochemical characterization of the encoded enzymes and the analysis of their terpene products, we wanted to learn about possible correlations between the catalytic activity and the structure of the active center of the enzyme. A terpene synthase with an especially complex reaction mechanism is TPS4 which produces a mixture of 14 different olefinic sesquiterpenes. To understand the complex TPS4 reaction mechanism, we modeled the active site cavity and conducted docking simulations with the substrate farnesyl diphosphate (FPP), several predicted carbocation intermediates, and the final reaction products. The model suggests that discrete steps of the reaction sequence are controlled by two different active site pockets, with the conformational change of the bisabolyl cation intermediate causing a shift from one pocket to the other. Site-directed mutagenesis and measurements of mutant activity in the presence of (E,E)- and (Z,E)-farnesyl diphosphate as substrates were employed to test this model. Amino acid alterations in pocket I indicated that early steps of the catalytic process up to the formation of the monocyclic bisabolyl cation are probably localized in this compartment. Mutations in pocket II primarily inhibited the formation of bicylic compounds, suggesting that secondary cyclizations of the bisabolyl cation are catalyzed in pocket II.
The catalytic structures responsible for the wide range of product specificity among the terpene synthases were studied by an extensive mutational analysis of the distantly related genes tps4 and tps10. Alteration of the amino acids forming the active site cavities were sufficient to turn the product specificity of TPS4 essentially into that of TPS10, indicating that only a small number of amino acids provides a scaffold for the complex reaction mechanism and thereby determines the product specificity of these terpene synthases.
Most of the terpenes emitted by maize after herbivore damage are products of the terpene synthase TPS10. These TPS10 volatiles attract natural enemies of the herbivores and thereby constitute an important defense signal of the plant. To elucidate the evolution of this plant defense signal and its biosynthesis, we study the herbivore-induced volatile emissions from several maize cultivars, teosinte, and other related grasses. The volatile signal is preserved throughout the grasses but shows slight differences in compositon in the different clades. Genes ortholog to tps10 are identified in several grasses to analyze the molecular evolution of tps10 and its defense trait which has been adopted at some point during the phylogeny of the plant.
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