Student Williams College Williamstown, Massachusetts
Body of Abstract: A variety of plant species including Zea mays (maize) and Vitis labrusca (fox grape) have convergently evolved the ability to synthesize O-methyl anthranilate from anthranilate, an intermediate in tryptophan biosynthesis. In maize, a single step reaction is catalyzed by a SAM-dependent anthranilate O-methyltransferase (AAMT1), however, in Concord grape, a two-step two-enzyme pathway is responsible for O-methyl anthranilate production. Some plants in the Citrus family (Rutaceae), like Citrus sinensis (sweet orange), produce both O-methyl anthranilate and N-methyl anthranilate, a compound with unknown in vivo function, using unidentified methyltransferases. Here, we aimed to biochemically trace the evolution of substrate specificity in ZmAAMT1. Additionally, we sought to identify and biochemically characterize the enzyme responsible for N-methyl anthranilate production in C. sinensis. Using a combination of phylogenetics, structural biology, and site-directed mutagenesis, we have successfully designed ZmAAMT1 mutants that introduced salicylic acid activity into ZmAAMT1; increased activity with anthranilate; and abolished activity with anthranilate entirely. In addition, we have successfully discovered and characterized the first anthranilate N-methyltransferase in C. sinensis CsANMT and identified enzymes capable of O-methyl anthranilate biosynthesis in C. sinensis. Overall, this work will improve our understanding of the evolution of anthranilate metabolism in plants and may help shed light on how primary and specialized anthranilate metabolism is regulated to optimize plant fitness.