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.     

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.     

Enzymatic preparation of metabolic intermediates, 3-dehydroquinate and 3-dehydroshikimate, in the shikimate pathway

A method for enzymatic preparation of 3-dehydroquinate and 3-dehydroshikimate in the shikimate pathway was established by controlling the enzyme activity of 3-dehydroquinate dehydratase. When quinate was incubated with the membrane fraction of acetic acid bacteria at pH 5.0, 3-dehydroquinate was formed as the predominant product. 3-Dehydroshikimate was the sole product when incubated at pH 8.0. Mutual separation of the metabolic intermediates was also exemplified.     

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.     

The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction.

The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.     

Phytotoxic and Metabolic Effects of Exogenous Quinate on Pisum sativum L.

Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side branch of the shikimate biosynthesis pathway. Plants treated with herbicides that inhibit amino acid biosynthesis (branched-chain and aromatic) accumulate quinate in their leaves. The objective of this study was to evaluate whether quinate mimics the effects of herbicides in plants. In pea plants, exogenous application of quinate through the nutrient solution was compared with leaf spraying at a concentration of 4 and 400 mM, respectively, and evaluated in parallel to the effects of herbicides. The analysis facilitated an assessment of the phytotoxicity and potential use of quinate as a natural herbicide. The application of quinate through the nutrient solution, but not the spray, was lethal, although both treatments affected plant growth. Quinate was absorbed and translocated to other plant organs remote from the application site, and an increase in the levels of aromatic amino acids and caffeic acid (that is, compounds located after quinate in the shikimate biosynthesis pathway) was detected, which indicates that quinate was metabolized and incorporated into the shikimate pathway. Exogenous application of quinate affected the carbohydrate content in the leaves and roots in a way similar to the toxic effects of herbicides. The phytotoxic effects of quinate reported in this study suggest that this compound deregulates the shikimate pathway and mimics some physiological effects described in the mode of action of herbicides inhibiting amino acid biosynthesis.     

Characterization of shikimate dehydrogenase homologues of Corynebacterium glutamicum

The function of three Corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsuD (cgR_0495), cgR_1216, and aroE (cgR_1677), was investigated. A disruptant of aroE required shikimate for growth, whereas a qsuD-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. There was no discernible difference in growth rate between wild-type and a cgR_1216-deficient strain. Enzymatic assays showed that AroE both reduced 3-dehydroshikimate, using NADPH as cofactor, and oxidized shikimate, the reverse reaction, using NADP⁺ as cofactor. The reduction reaction was ten times faster than the oxidation. QsuD reduced 3-dehydroquinate using NADH and oxidized quinate using NAD⁺ as cofactor. Different from the other two homologues, the product of cgR_1216 displayed considerably lower enzyme activity for both the reduction and the oxidation. The catalytic reaction of QsuD and AroE was highly susceptible to pH. Furthermore, reduction of 3-dehydroshikimate by AroE was inhibited by high concentrations of shikimate, but neither quinate nor aromatic amino acids had any effect on the reaction. Expression of qsuD mRNA was strongly enhanced in the presence of shikimate, whereas that of cgR_1216 and aroE decreased. We conclude that while AroE is the main catalyst for shikimate production in the shikimate pathway, QsuD is essential for quinate/shikimate utilization.     

High shikimate production from quinate with two enzymatic systems of acetic acid bacteria

3-Dehydroshikimate was formed with a yield of 57-77% from quinate via 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. 3-Dehydroshikimate was then reduced to shikimate (SKA) with NADP-dependent SKA dehydrogenase (SKDH) from the same organism. When SKDH was coupled with NADP-dependent D-glucose dehydrogenase (GDH) in the presence of excess D-glucose as an NADPH re-generating system, SKDH continued to produce SKA until 3-dehydroshikimate added initially in the reaction mixture was completely converted to SKA. Based on the data presented, a strategy for high SKA production was proposed.     

Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.     

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.     

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Biosynthesis of l-Phenylalanine and l-Tyrosine in the Actinomycete Amycolatopsis methanolica

Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.     

Gene-enzyme relationships of the arom aggregate of Schizosaccharomyces pombe

The gene-enzyme relationships of the arom multienzyme complex of Schizosaccharomyces pombe that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway have been studied. The various mutants were subjected to biochemical analysis by direct enzymic assays. These studies have established that aro-3A, aro-3B, aro-3C, aro-3D, and aro-3E mutants lack, respectively, the enzymic activities 5-dehydroquinate synthase, 5-dehydroquinase, shekimate kinase, 3-enolpyruvylshikimate 5-phosphate synthase, and shikimate: NADP oxidoreductase. In S. pombe lack enzymic activities for the inducible quinate catabolic pathway. The functional significance of the arom aggregate is discussed.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

L-Aspartate semialdehyde and a 6-deoxy-5-ketohexose 1-phosphate are the precursors to the aromatic amino acids in Methanocaldococcus jannaschii

No orthologs are present in the genomes of the archaea encoding genes for the first two steps in the biosynthesis of the aromatic amino acids leading to 3-dehydroquinate (DHQ). The absence of these genes prompted me to examine the nature of the reactions involved in the archaeal pathway leading to DHQ in Methanocaldococcus jannaschii. Here I report that 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde are precursors to DHQ. The sugar, which is derived from glucose 6-P, supplies a "hydroxyacetone" fragment, which, via a transaldolase reaction, undergoes an aldol condensation with the l-aspartate semialdehyde to form 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid. Despite the fact that both hydroxyacetone and hydroxyacetone-P were measured in the cell extracts and confirmed to arise from glucose 6-P, neither compound was found to serve as a precursor to DHQ. This amino sugar then undergoes a NAD dependent oxidative deamination to produce 3,7-dideoxy-d-threo-hept-2,6-diulosonic acid which cyclizes to 3-dehydroquinate. The protein product of the M. jannaschii MJ0400 gene catalyzes the transaldolase reaction and the protein product of the MJ1249 gene catalyzes the oxidative deamination and the cyclization reactions. The DHQ is readily converted into dehydroshikimate and shikimate in M. jannaschii cell extracts, consistent with the remaining steps and genes in the pathway being the same as in the established shikimate pathway.     

Purification and characterization of 3-dehydroquinate hydrolase and shikmate oxidoreductase. Evidence for a bifunctional enzyme

The basis for the physical association of 3-dehydroquinate dehydratase (3-dehydroquinate hydrolyase, EC 4.2.1.10) and shikimate dehydrogenase (shikimate: NADP+ 3-oxidoreductase, EC 1.1.1.25) in higher plants was investigated. The enzymes were extracted from the moss Physcomitrella patens and were purified to homogeneity. Determinations of subunit sizes were made by sodium dodecyl sulfate gel electrophoresis and gel exclusion chromatography in 6 M guanidinium chloride. Results from these studies demonstrate that both enzyme activities are carried out by a single polypeptide.     

Insights into the mechanism of type I dehydroquinate dehydratases from structures of reaction intermediates

The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.     

Enzymological basis for growth inhibition by L-phenylalanine in the cyanobacterium Synechocystis sp. 29108

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type.