Search for: All records

While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modu- lated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examina- tion identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants. 

Plants are often attacked by insects and other herbivores. As a result, they have evolved to defend themselves by producing many different chemicals that are toxic to these pests. As producing each chemical costs energy, individual plants often only produce one type of chemical that is targeted towards their main herbivore. Related species of plants often use the same type of chemical defense so, if a particular herbivore gains the ability to cope with this chemical, it may rapidly become an important pest for the whole plant family. To escape this threat, some plants have gained the ability to produce more than one type of chemical defense. Wallflowers, for example, are a group of plants in the mustard family that produce two types of toxic chemicals: mustard oils, which are common in most plants in this family; and cardenolides, which are an innovation of the wallflowers, and which are otherwise found only in distantly related plants such as foxglove and milkweed. The combination of these two chemical defenses within the same plant may have allowed the wallflowers to escape attacks from their main herbivores and may explain why the number of wallflower species rapidly increased within the last two million years. Züst et al. have now studied the diversity of mustard oils and cardenolides present in many different species of wallflower. This analysis revealed that almost all of the tested wallflower species produced high amounts of both chemical defenses, while only one species lacked the ability to produce cardenolides. The levels of mustard oils had no relation to the levels of cardenolides in the tested species, which suggests that the regulation of these two defenses is not linked. Furthermore, Züst et al. found that closely related wallflower species produced more similar cardenolides, but less similar mustard oils, to each other. This suggests that mustard oils and cardenolides have evolved independently in wallflowers and have distinct roles in the defense against different herbivores. The evolution of insect resistance to pesticides and other toxins is an important concern for agriculture. Applying multiple toxins to crops at the same time is an important strategy to slow the evolution of resistance in the pests. The findings of Züst et al. describe a system in which plants have naturally evolved an equivalent strategy to escape their main herbivores. Understanding how plants produce multiple chemical defenses, and the costs involved, may help efforts to breed crop species that are more resistant to herbivores and require fewer applications of pesticides. 

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products. 

Abstract Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys302 and Glu267 resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD+ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD+ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe169 with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae.