Molecular Basis of TyrA Substrate Specificity Underlying the Evolution of Alternative Tyrosine Biosynthetic Pathways

Abstract

An aromatic amino acid, tyrosine, is essential for protein synthesis in all orders of life and also serves as a precursor to numerous specialized metabolites crucial for plant and human health (e.g., vitamin E, isoquinoline alkaloids, and plastoquinone). Most plants synthesize tyrosine via arogenate dehydrogenase (ADH/TyrAa) that is strongly feedback inhibited by tyrosine, whereas we recently found that soybean and Medicago truncatula have, besides ADHs, prephenate dehydrogenase (PDH/TyrAp) enzymes, which are completely insensitive to tyrosine. However, molecular and evolutionary mechanisms underlying TyrA substrate specificity and the two alternative tyrosine pathways are currently unknown. In this study, we identified non‐canonical ADHs (ncADHs), which are phylogenetically very similar to PDHs, but prefer arogenate substrate and are partially inhibited by tyrosine. By combining the x‐ray crystal structure of soybean PDH (GmPDH) and the phylogenetic distribution of PDH enzymes, we identified putative substrate specificity determining residues in the recently diverged PDH and ncADH homologs. Site‐directed mutagenesis of Asn222 on GmPDH, which corresponds to an Asp in soybean ncADH (GmncADH) resulted in loss of PDH and gain of ADH activity. Consistently, the reciprocal mutation on GmncADH, D218N, introduced PDH at the expense of ADH activity. The introduction of an Asp residue on GmPDH also resulted in gain of tyrosine inhibition, which allowed co‐crystallization of the GmPDH N222D mutant with tyrosine in the active site and demonstrated that Asp222 directly interacts with the side chain amine of tyrosine and likely arogenate. Furthermore, mutating the corresponding Asp on divergent plant ADHs resulted in a gain of PDH activity and relaxation of tyrosine inhibition. Thus, the corresponding Asn of PDHs and Asp of ADHs simultaneously control substrate specificity and tyrosine regulation, which can be altered in various plants to enhance the production of tyrosine and its derived plant natural products.