Biochemical investigation of the tryptophan biosynthetic enzyme anthranilate phosphoribosyltransferase in plants.

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 modulated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examination identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants.

A structure-guided computational screening approach for predicting plant enzyme-metabolite interactions.

Plants are molecular factories that have spent millions of years evolving the enzymes needed to synthesize diverse primary and specialized metabolites. Despite the wealth of metabolites that plants produce, many of the enzymes responsible for generating these molecules have yet to be identified. For enzymes with known substrates, the extent of substrate promiscuity and small-molecule regulation remains unexplored. Many computational methods for identifying metabolic enzymes focus on gene-based approaches that rely on transcriptomics, metabolomics, and comparative genomics. With new AI-based tools for accurate protein structure prediction, protein-based strategies that screen a library of small molecules against a high-quality protein model can facilitate the identification of substrates, products, or inhibitors. Virtual screening has been used for structure-based drug design in the pharmaceutical industry for decades and easily translates to investigating plant metabolic enzymes. Here, we present a method for rapid, user-friendly, and open-source virtual screening using the Arabidopsis thaliana UGT74F2 with a curated library of specialized metabolites and herbicides and AutoDock Vina as an example. This method may be applied broadly to metabolic enzymes, and compound libraries can be easily adapted. Compounds are ranked based on their relative binding affinities and the resulting binding modes are evaluated using a molecular visualization program, like PyMOL. Because this is a computational approach, results from the virtual screen will need to be validated using in vitro or in vivo activity, binding, or inhibition assays. Virtual screening may aid in identifying substrates for enzymes of unknown function, revisiting substrate selectivity, or identifying natural or synthetic inhibitors.

Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.

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.

Investigating the reaction and substrate preference of indole-3-acetaldehyde dehydrogenase from the plant pathogen Pseudomonas syringae PtoDC3000.

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.

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

Independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae).

Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.

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.

Natural variation in the expression and catalytic activity of a naringenin 7-O-methyltransferase influences antifungal defenses in diverse rice cultivars.

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.

Brassicaceae-specific Gretchen Hagen 3 acyl acid amido synthetases conjugate amino acids to chorismate, a precursor of aromatic amino acids and salicylic acid.

To modulate responses to developmental or environmental cues, plants use Gretchen Hagen 3 (GH3) acyl acid amido synthetases to conjugate an amino acid to a plant hormone, a reaction that regulates free hormone concentration and downstream responses. The model plant Arabidopsis thaliana has 19 GH3 proteins, of which 8 have confirmed biochemical functions. One Brassicaceae-specific clade of GH3 proteins was predicted to use benzoate as a substrate and includes AtGH3.7 and AtGH3.12/PBS3. Previously identified as a 4-hydroxybenzoic acid-glutamate synthetase, AtGH3.12/PBS3 influences pathogen defense responses through salicylic acid. Recent work has shown that AtGH3.12/PBS3 uses isochorismate as a substrate, forming an isochorismate-glutamate conjugate that converts into salicylic acid. Here, we show that AtGH3.7 and AtGH3.12/PBS3 can also conjugate chorismate to cysteine and glutamate, which act as precursors to aromatic amino acids and salicylic acid, respectively. The X-ray crystal structure of AtGH3.12/PBS3 in complex with AMP and chorismate at 1.94 Å resolution, along with site-directed mutagenesis, revealed how the active site potentially accommodates this substrate. Examination of Arabidopsis knockout lines indicated that the gh3.7 mutants do not alter growth and showed no increased susceptibility to the pathogen Pseudomonas syringae, unlike gh3.12 mutants, which were more susceptible than WT plants, as was the gh3.7/gh3.12 double mutant. The findings of our study suggest that GH3 proteins can use metabolic precursors of aromatic amino acids as substrates.

Arabidopsis: the original plant chassis organism.

Arabidopsis thaliana (thale cress) has a past, current, and future role in the era of synthetic biology. Arabidopsis is one of the most well-studied plants with a wealth of genomics, genetics, and biochemical resources available for the metabolic engineer and synthetic biologist. Here we discuss the tools and resources that enable the identification of target genes and pathways in Arabidopsis and heterologous expression in this model plant. While there are numerous examples of engineering Arabidopsis for decreased lignin, increased seed oil, increased vitamins, and environmental remediation, this plant has provided biochemical tools for introducing Arabidopsis genes, pathways, and/or regulatory elements into other plants and microorganisms. Arabidopsis is not a vegetative or oilseed crop, but it is as an excellent model chassis for proof-of-concept metabolic engineering and synthetic biology experiments in plants.

Reaction Mechanism of Prephenate Dehydrogenase from the Alternative Tyrosine Biosynthesis Pathway in Plants.

Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine-insensitive biosynthesis of 4-hydroxyphenylpyruvate, a precursor to tyrosine. The three-dimensional structure of soybean PDH1 was recently solved in complex with the NADP+ cofactor. This structure allowed for the identification of both the cofactor- and ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active-site mutants of soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.

Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis.

Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X-ray crystal structures of the wild-type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady-state kinetic and ligand-binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co-crystallized with either α-ketoglutarate or pyridoxamine 5'-phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.

Evolution of allosteric regulation in chorismate mutases from early plants.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 ŠX-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.

L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

Dissonance Strikes a Chord in Stilbene Synthesizers.

In this issue of Cell Chemical Biology, Mori et al. (2016) combine X-ray crystallography and biochemistry to discover a new mechanism for stilbene synthesis in bacteria. The dialkyl-condensing enzyme StlD catalyzes formation of cyclohexanediones using a non-canonical ß-ketosynthase active site. Aromatization by StlC completes production of the stilbene product.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.