Evolution of substrate specificity in a maize anthranilate methyltransferase

Abstract

Secondary metabolites play a key role in mediating pollination and plant defense against herbivory. One such metabolite, methyl anthranilate, is a volatile compound responsible for the recognizable scent of Concord grapes. Methyl anthranilate is commercially used as a bird repellent for golf courses and as a flavoring agent for grape‐flavored foods, beverages, and pharmaceuticals. A variety of plant species such as Zea mays (maize) and Vitis labrusca (fox grape) synthesize methyl anthranilate from anthranilate, an intermediate in tryptophan biosynthesis. Methyl anthranilate biosynthesis is a classic example of convergent evolution; in maize, this reaction is catalyzed by an anthranilate methyltransferase (AAMT1), while in grape, there is a two‐step pathway. Previous studies have found that the ZmAAMT1 SABATH methyltransferase likely evolved from methyltransferases that recognize the plant pathogen response hormone salicylic acid as a substrate. Using site‐directed mutagenesis, we introduced 12 independent missense mutations at 7 key active site residues with the intention of restoring substrate specificity for salicylic acid. This work will allow us to better trace and understand the evolution of anthranilate‐derived specialized metabolism in plants.