Substrates, enzymes, and products of the shikimate pathway.
Phenolic compounds are secondary metabolites found most abundantly in plants. These aromatic molecules have important roles, as pigments, antioxidants, signaling agents, the structural element lignan, and as a defense mechanism. The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). These compounds are formed via the shikimate pathway in higher plants and microorganisms. The enzymes responsible for the regulation of phenolic metabolism are known, and shikimic acid is a central metabolite. The shikimate pathway consists of seven reaction steps, beginning with an aldol-type condensation of phosphoenolpyruvic acid (PEP) from the glycolytic pathway, and D-erythrose-4-phosphate, from the pentose phosphate cycle, to produce 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP). A key branch-point compound is chorismic acid, the final product of the shikimate pathway. The shikimate pathway is described in this chapter, as well as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the production of phenolic compounds in plants are discussed.
- shikimate pathway
- phenolic compounds
- biosynthetic routes
- phenylpropanoid metabolism
The secondary metabolism is a biosynthetic source of several interesting compounds useful to chemical, food, agronomic, cosmetics, and pharmaceutical industries. The secondary pathways are not necessary for the survival of individual cells but benefit the plant as a whole . Another general characteristic of secondary metabolism is that found in a specific organism, or groups of organisms, and is an expression of the individuality of species . The secondary metabolism provides chemical diversity to organic molecules with low molecular weight that are related by the respective pathways; such organic molecules are called secondary metabolites. The secondary metabolites are often less than 1% of the total carbon in plant molecules . These organic molecules isolated from terrestrial plants are the most studied, and their syntheses have an important role in the protection against pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation . Secondary metabolism reflects plant environments more closely than primary metabolism . There are three principal kinds of secondary metabolites biosynthesized by plants: phenolic compounds, terpenoids/isoprenoids, and alkaloids and glucosinolates (nitrogen- or sulfur-containing molecules, respectively) . Phenolic compounds are biosynthesized by the shikimate pathway and are abundant in plants. The shikimate pathway, in plants, is localized in the chloroplast. These aromatic molecules have important roles, as pigments, antioxidants, signaling agents, electron transport, communication, the structural element lignan, and as a defense mechanism , Figure 1. The seven steps of the shikimate pathway and the metabolites for branch point are described in this chapter, as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the production of phenolic compounds in plants are discussed.
2. The shikimate pathway
The shikimate biosynthesis pathway provides precursors for aromatic molecules in bacteria, fungi, apicomplexan, and plants, but not in animals [2, 7]. Shikimic acid is named after the highly toxic Japanese
The shikimate pathway consists of seven sequential enzymatic steps and begins with an aldol-type condensation of two phosphorylated active compounds, the phosphoenolpyruvic acid (PEP), from the glycolytic pathway, and the carbohydrate D-erythrose-4-phosphate, from the pentose phosphate cycle, to give 3-deoxy-D-
|1||Phosphoenolpyruvate (PEP), erythrose-4-phosphate||3-Deoxy-D-
||3-Dehydroquinate synthase DHQS (EC. 220.127.116.11)/Co2+, NAD+ [15, 16]||3-Dehydroquinic acid (DHQ), Pi|
|3||3-Dehydroquinic acid (DHQ)||3-Dehydroquinate dehydratase (DHQ dehydratase EC 18.104.22.168) ||3-Dehydroshikimic acid (DHS), H2O|
|4||3-Dehydroshikimic acid (DHS), NADPH + H+||Shikimate dehydrogenase (SDH; EC 22.214.171.124) [18, 19, 20, 21]||Shikimic acid, NADP+|
|5||Shikimic acid, ATP||Shikimate kinase enzyme (SK; EC 126.96.36.199)||Shikimic acid 3-phosphate (S3P), ADP|
|6||Shikimic acid 3-phosphate (S3P), PEP||5-
||Chorismate synthase (CS; EC 188.8.131.52)/FMNH2 [2, 19, 30, 31]||Chorismic acid, Pi|
The shikimate pathway has special characteristics that are present only in bacteria, fungi, and plants. The absence of the pathway in all other organisms provides the enzymes catalyzing these reactions with potentially useful targets for the development of antibacterial agents and herbicides. For example, 5-
In the second reaction step, DAHP loses phosphate (Pi); the enolic-type product is cyclized through a second aldol-type reaction to produce 3-dehydroquinic acid (DHQ). The 3-dehydroquinate synthase (DHQS) catalyzes this cyclization in the shikimate pathway. The DHQ dehydrates to produce 3-dehydroshikimic acid (DHS) (3-dehydroquinate dehydratase); this compound has a conjugated double carbon-carbon, Figure 3. The protocatechuic and the gallic acids (C6-C1) are produced by branch-point reactions from DHS . The fourth step in the pathway is a reduction reaction of DHS with reduced nicotinamide adenine dinucleotide phosphate (NADPH), Figure 3. The fifth section of the pathway is the activation of shikimic acid with adenosine triphosphate (ATP) (shikimate kinase, SK) to make shikimic acid 3-phosphate (S3P). The sixth chemical reaction is the addition of PEP to S3P to generate 5-
The last reaction step of the shikimate pathway is the production of chorismic acid from catalytic action on the chorismate synthase (CS). This reaction is a 1,4-
2.1. Synthesis of 3-deoxy-D-
arabino-heptulosonic acid 7-phosphate (DAHP)
The first reaction of the shikimate pathway is an aldol-type condensation of PEP and carbohydrate erythrose-4-P, to give 3-deoxy-D-
2.2. Synthesis of 3-dehydroquinic acid (DHQ)
The second reaction of the shikimate pathway is an intramolecular aldol-type reaction cyclization, where the enol (C6-C7) of DAHP nucleophilically attacks the carbonyl group (C2), to produce a six-member cycle, the 3-dehydroquinic acid (DHQ), Figures 3 and 6. The enzyme that catalyzes this reaction, 3-dehydroquinate synthase DHQS (EC. 184.108.40.206), is a carbon-oxygen lyase enzyme that requires Co2+ and bound oxidized nicotinamide adenine dinucleotide (NAD+) as cofactors [15, 16]. The Co2+ is essential for the catalytic activity of DHQS. Bender et al.  found that DHQS, from
The reduction reaction of DHQ leads to quinic acid at this branch point in the shikimate pathway. Quinic acid is a secondary metabolite that is free, forming esters or as part of alkaloids such as quinine. Quinic acid is found in high quantities in mature kiwi fruit (
2.3. Synthesis of 3-dehydroshikimic acid (DHS) and shikimic acid
The third and fourth reaction steps of the shikimate pathway are catalyzed by a bifunctional enzyme: 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ dehydratase/SDH; EC 220.127.116.11/EC 18.104.22.168). The DHQ dehydratase enzyme is a hydro-lyase kind, and the SDH is an oxidoreductase enzyme. The DHQ dehydratase, in the third reaction step, converts DHQ into 3-dehydroshikimic acid (DHS) by eliminating water, and this reaction is reversible, Figure 7. The DHS is converted to shikimic acid in the fourth reaction step, by the reduction of the carbonyl group at C-5 by the catalytic action of SDH with NADPH, Figure 3. The biosynthesis of DHS is a branch point to shikimic acid and to the catabolic quinate pathway. If the DHS dehydrates, it produces protocatechuic acid (C6-C1) or gallic acid, Figure 3. Gallic acid (C6-C1) is a hydroxybenzoic acid that is a component of tannins .
Two structurally different kinds of 3-dehydroquinate dehydratase are known: type I (not heat-stable) and type II (heat-stable). Type I enzyme is present in bacteria and higher plants, and type II is found in fungi, which have both types of enzymes [18, 19]. The catalytic mechanism of the type I DHQ dehydratase has been detected by electrospray MS . This catalytic mechanism involves the amino acid residue Lys-241 that forms a Schiff base with the substrate and product, Figure 7 . The fourth step is the reduction of DHS with NADPH that enantioselectively reduces the carbonyl of the ketone group of DHS to produce shikimic acid (shikimate dehydrogenase, SDH), Figure 3.
Sigh and Christendat  reported the crystal structure of DHQ dehydratase/SDH from the plant genus
2.4. Synthesis of shikimic acid 3-phosphate (S3P)
The shikimate kinase enzyme (SK; EC 22.214.171.124) catalyzes the phosphorylation of the shikimic acid, the fifth chemical reaction of the shikimate pathway, and the products are shikimic acid 3-phosphate (S3P) and ADP, Figures 3 and 8. Shikimic acid is phosphorylated with ATP in the 5-hydroxyl group of shikimic acid. SK is an essential enzyme in several bacterial pathogens and is not present in the human cell; therefore the SK enzyme has been classified as a protein target for drug design, especially for chemotherapeutic development of antitubercular drugs [23, 24].
2.5. Synthesis of 5-
enolpyruvylshikimate 3-phosphate (EPSP)
EPSPS is the most studied enzyme of the shikimate pathway because it plays a crucial role in the penultimate step. If this enzyme is inhibited, there is an accumulation of shikimic acid , and the synthesis of aromatic amino acid is disabled, leading to the death of the plant . Therefore, EPSPS is used as a target for pesticides, like glyphosate, Figure 4, the active ingredient in the herbicides RoundUp™, Monsanto Chemical Co., and Touchdown™, Syngenta. Glyphosate (
2.6. Synthesis of chorismic acid
The seventh and last reaction step of the shikimate pathway is the 1,4-
3. Factors that induce the synthesis of phenolic compounds in plants
The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). UV radiation is divided into UVC (≤280 nm), UVB (280–320 nm), and UVA (300–400 nm). UVA and UVB radiation are transmitted through the atmosphere; all UVC and some UVB radiation (highly energetic) are absorbed by the Earth’s ozone layer. This accumulation is explained by the increase in enzymatic activity of the phenylalanine ammonia-lyase and chalcone synthase enzymes, among others . Studies have been done about the increase of phenolic compounds, such as anthocyanins, in plants when they are exposed to UVB radiation . Another study demonstrates that UVB exposure enhances anthocyanin biosynthesis in “Cripps pink” apples (
The increase in phenolic compounds in blueberry (
An interesting study was carried out in 2011 by Mody et al., where they studied the effect of the resistance response of apple tree seedlings (
Knowledge of the biosynthetic pathway of shikimic acid leads to understanding the reaction mechanisms of enzymes and thus discovering antimicrobials, pesticides, and antifungals. Studies with isotopic labeling of substrates, the use of X-ray diffraction, nuclear magnetic resonance (NMR), mass spectrometry (ES), biotechnology, as well as organic synthesis have contributed to explaining the shikimate pathway. Although the seven steps of the biosynthetic pathway are elucidated, these metabolites are the precursors of phenolic compounds, more complex molecules that are necessary for the adaptation of plants to the environment. So, the shikimate pathway is the basis for the subsequent biosynthesis of phenolic compounds. There is scientific interest in continuing to investigate the biosynthesis of phenolic compounds from several points of view: pharmaceuticals, agronomy, chemical and food industries, genetics, and health.
The authors thank Carol Ann Hayenga for her English assistance in the preparation of this manuscript. The Technological University of the Mixteca provided support.
Conflict of interest
The authors have no conflict of interest to declare and are responsible for the content and writing of the manuscript.
This chapter does not contain any studies with human participants or animals performed by any of the authors.
Adams ZP, Ehlting J, Edwards R. The regulatory role of shikimate in plant phenylalanine metabolism. Journal of Theoretical Biology. 2019; 462:158-170. DOI: 10.1016/j.jtbi.2018.11.005
Dewick PM. Medicinal Natural Products: A Biosynthetic Approach. 3rd ed. United Kingdom: John Wiley and Sons Ltd.; 2009. p. 539. DOI: 10.1002/9780470742761
Bourgaud F, Gravot A, Milesi S, Gontier E. Production of plant secondary metabolites: A historical perspective. Plant Science. 2001; 161:839-851. DOI: 10.1016/S0168-9452(01)00490-3
Yang D, Huang Z, Jin W, Xia P, Jia Q, Yang Z, et al. DNA methylation: A new regulator of phenolic acids biosynthesis in Salvia miltiorrhiza. Industrial Crops and Products. 2018; 124:402-411. DOI: 10.1016/j.indcrop.2018.07046
Aharoni A, Galili G. Metabolic engineering of the plant primary-secondary metabolism interface. Current Opinion in Biotechnology. 2011; 22:239-244. DOI: 10.1016/j.copbio.2010.11.004
Macheroux P, Schmid J, Amrhein N, Schaller A. A unique reaction in a common pathway: Mechanism and function of chorismate synthase in the shikimate pathway. Planta. 1999; 207:325-334
Mittelstädt G, Negron L, Schofiel LR, Marsh K, Parker EJ. Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis. Archives of Biochemistry and Biophysics. 2013; 537:185-191. DOI: 10.1016/j.abb.2013.07.022
Ghosh S, Chisti Y, Banerjee UC. Production of shikimic acid. Biotechnology Advances. 2012; 30:1425-1431. DOI: 10.1016/j.biotechadv.2012.03.001
Weaver LM, Herrmann KM. Dynamic of the shikimate pathway. Trends in Plant Science. 1997; 9:346-351
Tzin V, Galili G. Amino acids biosynthesis pathways in plants. Molecular Plant. 2010; 3:956-972. DOI: 10.1093/mp/ssq048
Dixon RA, Strack D. Phytochemistry meets genome analysis, and beyond. Phytochemistry. 2003; 62:815-816
Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S. Plant phenolics: Recent advances on their biosynthesis, genetics and ecophysiology. Plant Physiology and Biochemistry. 2013; 72:1-20. DOI: 10.1016/j.plaphy.2013.05.009
Zhang Z-Z, Li X-X, Chu Y-N, Zhang M-X, Wen Y-Q, Duan C-Q, et al. Three types of ultraviolet irradiation differentially promote expression of shikimate pathway genes and production of anthocyanins in grape berries. Plant Physiology and Biochemistry. 2012; 57:74-83. DOI: 10.1016/j.plaphy.2012.05.005
Floss HG, Onderka DK, Carroll M. Stereochemistry of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase reaction and the chorismite synthetase reaction. The Journal of Biological Chemistry. 1972; 247:736-744
Schmid J, Amrhein N. Molecular organization of the shikimate pathway in higher plants. Phytochemistry. 1995; 39:737-749
Bender SL, Mehdi S, Knowles JR. Dehydroquinate synthase: The role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis. Biochemistry. 1989; 28:7555-7560
Scholz BM, Maier G. Isomers of quinic acid and quinide in roasted coffee. Zeitschrift für Lebensmittel-Untersuchung und -Forschung. 1990; 190:132-134
Harris JM, Gonzalez-Bello C, Kleanthous C, Hawkins A, Coggins J, Abell C. Evidence from kinetic isotope studies for an enolate intermediate in the mechanism of type II dehydroquinases. Biochemical Journal. 1999; 319:333-336
Hermann KM. The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. The Plant Cell. 1995; 7:907-919
Shneier A, Kleanthous C, Deka R, Coggins JR, Abel C. Observation of an imine intermediate on dehydroquinase by electrospray mass spectrometry. Journal of the American Chemical Society. 1991; 113:9416-9418
Sigh SA, Christendat D. The DHQ-dehydroshikimate-SDH-shikimate-NADP(H) complex: Insights into metabolite transfer in the shikimate pathway. Crystal Growth & Design. 2007; 7:2153-2160
Sigh SA, Christendat D. Structure of Arabidopsisdehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway. Biochemistry. 2006; 45:7787-7796. DOI: 10.1021/bi060366+
Coracini JD, de Azevedo WF Jr. Shikimate kinase, a protein target for drug design. Current Medicinal Chemistry. 2014; 21:592-604. DOI: 10.2174/09298673113206660299
Blanco B, Prado V, Lence E, Otero JM, García-Doval C, van Raaij MJ, et al. Mycobacterium tuberculosisshikimate kinase inhibitors: Design and simulation studies of the catalytic turnover. Journal of the American Chemical Society. 2013; 135:12366-12376. DOI: 10.1021/ja405853p
Lewis J, Johnson KA, Anderson KS. The catalytic mechanism of EPSP synthase revisited. Biochemistry. 1999:7372-7379
Maroli A, Nandula V, Duke S, Tharayil N. Stable isotope resolved metabolomics reveals the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in Amaranthus palmeribiotypes. Journal of Agricultural and Food Chemistry. 2016; 64:7040-7048. DOI: 10.1021/acs.jafc.6b02196
Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, et al. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coliand Tobacco Plants. PLoS One. 2012; 7:e38718. DOI: 10.1371/journal.pone.0038718
Yi S-y, Cui Y, Zhao Y, Z-d L, Y-j L, Zhou F. A novel naturally occurring class I 5-enolpyruvyl shikimate-3-phosphate synthase from Janibactersp. confers high glyphosate tolerance to rice. Scientific Reports. 2016; 6:1904
Liu F, Cao Y. Expression of a bacterial aroA gene confers tolerance to glyphosate in tobacco plants. Turkish Journal of Biology. 2018; 42:187-194. DOI: 10.3906/biy-1712-56
Bornemann S, Theoclitou M-E, Brune M, Webb MR, Thorneley RNF, Abell C. A secondary β deuterium kinetic isotope effect in the chorismate synthase reaction. Bioorganic Chemistry. 2000; 28:191-204. DOI: 10.1006/bioo.2000.1174
Osborne A, Thorneley RNF, Abell C, Bornemann S. Studies with substrate and cofactor analogues provide evidence for radical mechanism in the chorismate synthase reaction. The Journal of Biological Chemistry. 2000; 275:35825-35830
Marais E, Jacobs G, Holcroft DM. Postharvest irradiation enhances anthocyanin synthesis in apples but nor in pears. HortScience. 2001; 36:738-740
Manquián-Cerda K, Cruces E, Escudey M, Zúñiga G, Calderón R. Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry ( Vaccinium corymbosumL.) plantlets cultivated in vitro. Ecotoxicology and Environmental Safety. 2018; 150:320-326. DOI: 10.1016/j.ecoenv.2017.12.050
Gutbrod B, Mody K, Wittwer R, Dorn S. Within-plant distribution of induced resistance in apple seedlings: Rapid acropetal and delayed basipetal responses. Planta. 2011; 233:1199-1207