Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants

The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non‐seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate‐specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.     

Molecular Characterization of Quinate and Shikimate Metabolism in Populus trichocarpa

The shikimate pathway leads to the biosynthesis of aromatic amino acids essential for protein biosynthesis and the production of a wide array of plant secondary metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single step reaction from 3-dehydroquinate catalyzed by quinate dehydrogenase (QDH). 3-Dehydroquinate is also the substrate for shikimate biosynthesis through the sequential actions of dehydroquinate dehydratase (DQD) and shikimate dehydrogenase (SDH) contained in a single protein in plants. The reaction mechanism of QDH resembles that of SDH. The poplar genome encodes five DQD/SDH-like genes (Poptr1 to Poptr5), which have diverged into two distinct groups based on sequence analysis and protein structure prediction. In vitro biochemical assays proved that Poptr1 and -5 are true DQD/SDHs, whereas Poptr2 and -3 instead have QDH activity with only residual DQD/SDH activity. Poplar DQD/SDHs have distinct expression profiles suggesting separate roles in protein and lignin biosynthesis. Also, the QDH genes are differentially expressed. In summary, quinate (secondary metabolism) and shikimate (primary metabolism) metabolic activities are encoded by distinct members of the same gene family, each having different physiological functions.     

3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.     

Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots

The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis‐inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched‐chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less‐efficient, ATP‐producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.     

Impairment of carbon metabolism induced by the herbicide glyphosate

The herbicide glyphosate reduces plant growth and causes plant death by inhibiting the biosynthesis of aromatic amino acids. The objective of this work was to determine whether glyphosate-treated plants show a carbon metabolism pattern comparable to that of plants treated with herbicides that inhibit branched-chain amino acid biosynthesis. Glyphosate-treated plants showed impaired carbon metabolism with an accumulation of carbohydrates in the leaves and roots. The growth inhibition detected after glyphosate treatment suggested impaired metabolism that impedes the utilization of available carbohydrates or energy at the expected rate. These effects were common to both types of amino acid biosynthesis inhibitors. Under aerobic conditions, ethanolic fermentative metabolism was enhanced in the roots of glyphosate-treated plants. This fermentative response was not related to changes in the respiratory rate or to a limitation of the energy charge. This response, which was similar for both types of herbicides, might be considered a general response to stress conditions.     

Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases

Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH‐specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase–shikimate dehydrogenases (DHQD‐SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD⁺ and NADP⁺ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development.     

Molecular organization of the shikimate pathway in higher plants

The shikimate pathway produces the three proteinogenic aromatic amino acids, phenylalanine, tyrosine and tryptophan, which are, in addition to several intermediates of the shikimate pathway, intermediates in the biosynthesis of numerous aromatic natural products in higher plants. While there is only little difference in the sequence of the chemical reactions of the pathway in bacteria, fungi and plants, considerable differences exist in the organization and regulation of the shikimate pathway in plants, fungi and bacteria. The recent isolation and characterization of cDNAs and genes coding for enzymes of the shikimate pathway in higher plants have confirmed that plastids are the major, if not only site of aromatic amino acid biosynthesis in plants. Furthermore, the observed differential spatial and temporal expression of genes coding for isozymes of the pathway indicates a complex regulation that we are only beginning to understand.     

In vitro ruminal fermentation of organic acids common in forage.

Mixed rumen bacteria from cows fed either timothy hay or a 60% concentrate were incubated with 7.5 mM citrate, trans-aconitate, malate, malonate, quinate, and shikimate. Citrate, trans-aconitate, and malate were fermented at faster rates than malonate, quinate, and shikimate. Acetate was the primary fermentation product for all six acids. Quinate and shikimate fermentations gave rist to butyrate, whereas malate and malonate produced significant amounts of propionic acid. High-pressure liquid chromatography of fermentation products from trans-aconitate incubations revealed a compound that was subsequently identified as tricarballylate. As much as 40% of the trans-aconitate acid was converted to tricarballylate, and tricarballylate was fermented slowly. The slow rate of tricarballylate metabolism by mixed rumen bacteria and its potential as a magnesium chelator suggest that tricarballylate formation could be an important factor in the hypomagnesemia that leads to grass tetany.     

Structures of shikimate dehydrogenase AroE and its Paralog YdiB. A common structural framework for different activities

Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar architecture with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.     

Purification,cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.     

Functional analysis of the essential bifunctional tobacco enzyme 3-dehydroquinate dehydratase/shikimate dehydrogenase in transgenic tobacco plants

In plants, the shikimate pathway occurs in the plastid and leads to the biosynthesis of aromatic amino acids. The bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SHD) catalyses the conversion of dehydroquinate into shikimate. Expression of NtDHD/SHD was suppressed by RNAi in transgenic tobacco plants. Transgenic lines with <40% of wild-type activity displayed severe growth retardation and reduced content of aromatic amino acids and downstream products such as cholorogenic acid and lignin. Dehydroquinate, the substrate of the enzyme, accumulated. However, unexpectedly, so did the product, shikimate. To exclude that this finding is due to developmental differences between wild-type and transgenic plants, the RNAi approach was additionally carried out using a chemically inducible promoter. This approach revealed that the accumulation of shikimate was a direct effect of the reduced activity of NtDHD/SHD with a gradual accumulation of both dehydroquinate and shikimate following induction of gene silencing. As an explanation for these findings the existence of a parallel extra-plastidic shikimate pathway into which dehydroquinate is diverted is proposed. Consistent with this notion was the identification of a second DHD/SHD gene in tobacco (NtDHD/SHD-2) that lacked a plastidic targeting sequence. Expression of an NtDHD/SHD-2-GFP fusion revealed that the NtDHD/SHD-2 protein is exclusively cytosolic and is capable of shikimate biosynthesis. However, given the fact that this cytosolic shikimate synthesis cannot complement loss of the plastidial pathway it appears likely that the role of the cytosolic DHD/SHD in vivo is different from that of the plastidial enzyme. These data are discussed in the context of current models of plant intermediary metabolism.     

Non-light-dependent Shikimate Pathway in Plastids from Pea Roots

Non-green plastids (leucoplasts) isolated from pea roots are shown to be considerably active in forming aromatic amino acids by the shikimate pathway which, in contrast to the chloroplast pathway, is independent of light. Supply of phosphoenolpyruvate and 3-dehydroquinate, 3-dehydroshikimate, shikimate and quinate effectively enhances the formation of aromatic amino acids suggesting an intra- or/and intercellular intermediate transport.     

The Metabolism of Quinate in Pea Roots (Purification and Partial Characterization of a Quinate Hydrolyase)

A quinate (QA) hydrolyase was isolated from pea (Pisum sativum L.) roots. The enzyme converts QA into shikimate by elimination of water. The enzymatic reaction is independent of cofactors and divalent cations. The QA hydrolyase was purified about 1,600-fold to apparent electrophoretic homogeneity in three steps, including bovine serum albumin-affinity chromatography. The enzyme forms oligomers and/or complexes with bovine serum albumin and ovalbumin. The monomer molecular weight of the enzyme is about 15,000. The hydrolyase shows regular Michaelis-Menten kinetics with a Km, of 2.0 mM for QA. Compartmentation studies reveal that the QA hydrolyase is localized in plastids. The QA hydrolyase may function in channeling imported QA into the shikimate pathway to support aromatic amino acid biosynthesis in plastids.     

Hydroaromatic metabolism in Rhodococcus rhodochrous: purification and characterisation of its NAD-dependent quinate dehydrogenase

Quinate grown cells of Rhodococcus rhodochrous N75 metabolized both quinate and shikimate via protocatechuate to succinate and acetyl CoA. The initial enzyme of the hydroaromatic pathway, quinate dehydrogenase was purified 188-fold to electrophoretic homogeneity. The enzyme is a monomer with a native relative molecular mass of 44,000 and is NAD-dependent. The enzyme is highly stereospecific with regard to hydroaromatic substrates, oxidising only the axial hydroxyl group at C-3 of (-)-isomers of quinate, shikimate, dihydroshikimate and t-3,t-4-dihydroxycyclohexane-c-1-carboxylate, but shows activity with several NAD analogues.     

The use of mutants and transgenic plants to study amino acid metabolism

Mutants of higher plants with alterations in amino acid metabolism have now been available for 20 years. Following the realization that at least four distinct classes of herbicides (phosphinothricins, glyphosates, imidazolinones and sulphonylureas) act by the inhibition of amino acid biosynthesis, mutants resistant to the herbicides have also been obtained. More recently, transgenic plants containing altered levels of enzymes of amino acid biosynthesis have been constructed. In this article, we have attempted to review several areas of amino acid biosynthesis including ammonia assimilation, the aspartate pathway, branched chain amino acids, aromatic amino acids and proline.     

Structural basis for shikimate-binding specificity of Helicobacter pylori shikimate kinase

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Angstroms crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel beta-strands flanked by seven alpha-helices. An HpSK-shikimate-PO(4) complex was also determined and refined to 2.3 Angstroms, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3(10) helix formed by a strictly conserved motif (GGGXV) after beta(3). Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.