Also, the quinic acid is abundant in roasted coffee [ 17 ]. The biosynthesis of DHS is a branch point to shikimic acid and to the catabolic quinate pathway. Gallic acid C6-C1 is a hydroxybenzoic acid that is a component of tannins [ 2 ]. Figure 7. 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 ].
This catalytic mechanism involves the amino acid residue Lys that forms a Schiff base with the substrate and product, Figure 7 [ 21 ]. 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 ].
Figure 8. Phosphorylation of shikimic acid with ATP. Figure 9. 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 [ 26 ], and the synthesis of aromatic amino acid is disabled, leading to the death of the plant [ 27 ].
Because the glyphosate is nonselective and kills food crops, there is interest in finding glyphosate-tolerant genes for genetically modified crops [ 29 ]. Synthesis of chorismic acid The seventh and last reaction step of the shikimate pathway is the 1,4-trans elimination of the Pi group at C-3 from EPSPS to synthetize chorismic acid.
This last step is catalyzed by chorismate synthase CS; EC 4. The FMNH2 transfers an electron to the substrate reversibly [ 30 ]. Spectroscopic techniques and kinetic isotope effect studies suggest that a radical intermediate in a non-concerted mechanism is developed [ 30 , 31 ], Figure Chorismic acid, the final molecule of the shikimate pathway, is a key branch point to post-chorismic acid pathways, to obtain L-Phe, L-Tyr, and L-Trp, Figure 2.
L-Phe is the substrate to phenylpropanoid and flavonoid pathways [ 13 ]. Figure Reaction of mechanism to yield chorismic acid by chorismate synthase . Factors that induce the synthesis of phenolic compounds in plants The expression of phenolic compounds is promoted by biotic and abiotic stresses e.
This accumulation is explained by the increase in enzymatic activity of the phenylalanine ammonia-lyase and chalcone synthase enzymes, among others [ 12 ]. Studies have been done about the increase of phenolic compounds, such as anthocyanins, in plants when they are exposed to UVB radiation [ 13 ]. This effect may be due to UV radiation exposure and the cultivar of the plants studied. It is known that if plants are under stress, they accumulate phenolic compounds.
These heavy metals cause high toxicity in plants, because they increase the oxidative stress by the production of reactive oxygen species ROS. The authors of the study suggest that the phenolic compounds, specifically chlorogenic and ellagic acids, Figure 11 , reduce the ROS in blueberry plants [ 33 ].
Chemical structure of chlorogenic C6-C3 and ellagic C6-C1 acids. Also, the results showed higher concentrations of the flavonoid phlorizin, Figure 12 , in damaged plants than undamaged plants. Bands were excised, washed in sterile water, and dried. Tryptic digestion was performed as described Lee et al. To clone SDH from A. PCR-based cloning was performed. SDH from walnut was obtained from juvenile seed coat of J. Degenerate primers were used to amplify the shikimate dehydrogenase message.
AroE, pAt. SDH for the E. Functional complementation in E. Electrochemically competent AB cells were generated and transformed with each expression vector construct as described Ausubel Vector construction for Agrobacterium-mediated plant transformation A full-length transit peptide from the Silene pratensis ferredoxin precursor, designated pTP12, was kindly provided by K. Inoue University of California, Davis Smeekens et al. The PCR conditions were as for J. Clone pTP. The SDH gene from pJr.
SDH was then ligated to the transit peptide to generate pDU This expression cassette was then ligated into the binary plasmid pDU As a negative control, the transit peptide from pDU The resultant binary vectors were transformed into the disarmed A. Plant Transformation The Ralph M. Transformations with these plasmids were confirmed through PCR. Overexpression of J. Results Co-localization of shikimic acid and gallic acid production in wild type plants Soluble protein isolated from wild type E.
Soluble protein from N. As expected, E. Of the J. Open image in new window Fig. SDH fror A. Protein from the following samples are in lanes m pJr. We detected SDH activity in a single section of each gel. Gallic acid production at a given SA concentration was highest in the two tree species, J. For each bacterial and plant sample tested, only protein from the region of the gel containing the SDH band catalyzed GA production. Bands were excised from the gel and tested for gallic acid production.
Gallic acid production was measured from each of the following: black inverted triangle purified Ec. AroE; green triangle purified Jr. SDH [ - substrate shikimic acid ]; orange square purified Jr. SDH denatured protein ; red circle purified Jr. SDH; blue diamond purified At. SDH and purple diamond purified protein from E. Conversely, wild-type E. Proteins isolated from wild type E. Conversely,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 the 3-dehydroquinic acid DHQFigures 3 and 6. However, GA biosynthesis in plants has not been investigated. Inoue University of California, Davis Smeekens et al. Two structurally different kinds of 3-dehydroquinate dehydratase are known: monitored as described in Gomez et al.
Wild type protein produced Conversely, wild-type E. Bands were excised, washed in sterile water, and dried. Electrochemically competent AB cells were generated and transformed with each expression vector construct as described Ausubel Collision energy for fragmentation used for MS2 experiments was set at 1.
This accumulation is explained by the increase in enzymatic activity of the phenylalanine ammonia-lyase and chalcone synthase enzymes, among others [ 12 ]. EPSPS is the most studied enzyme of the shikimate pathway because it plays a crucial role in the penultimate step. Grape berry growth is characterized by a double sigmoid curve Coombe and Hale,