Petroselinum crispum, SDH, SDH

Hýskova, Veronika, Belonozníkov, Katerina, Smeringaiova, Ingrida, Kavan, Daniel, Ingr, Marek & Ryslava, Helena, 2021, How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?, Phytochemistry (112881) 190, pp. 1-12 : 8-9

3.4. P. crispum root SDH is regulated by metabolites of the phenylpropanoid pathway

Because the shikimate pathway is absent in mammals, searching and designing inhibitors against enzymes of this pathway may lead to the development of antimicrobials (such as the bacterial Mycobacterium tuberculosis and Helicobacter pylori SDH ) and antiparasitic (malaria parasite SDH) and herbicidal (plant SDH) agents, which are harmless to humans ( Diaz-Quiroz et al., 2018). There are 3 strategies for identifying compounds with an inhibitory effect on a particular enzyme: i) analyzing substrate structural analogs ( Baillie et al., 1972; Diaz and Merino, 1997; Fiedler and Schultz, 1985; Koshiba, 1978; Lemos Silva et al., 1985; Lourenco and Neves, 1984; Lourenco et al., 1991; Rothe, 1974), ii) screening thousands of compounds ( Avitia-Dominguez et al., 2014; Han et al., 2006; Peek et al., 2014), and iii) searching for feedback inhibitors among products of the whole pathway. The first strategy has led to the discovery of the herbicide 2,4-dichlorphenoxy acetic acid (2, 4-D) ( Diaz and Merino, 1997). Concurrently, several studies have demonstrated that PCA (possible byproduct of SDH) inhibits plant SDH ( Diaz and Merino, 1997; Koshiba, 1978; Lemos Silva et al., 1985; Lourenco and Neves, 1984; Lourenco et al., 1991). In this study, we have shown that P. crispum SDH forms PCA in the irreversible reaction ( Fig. 4 ). Using a screening strategy, different research groups have identified SDH inhibitors, for example, 5 novel Helicobacter pylori SDH inhibitors, including the natural product curcumin ( Han et al., 2006), and polyphenolic inhibitors (epigallocatechin gallate and epicatechin gallate) of Pseudomonas putida and Arabidopsis thaliana SDH ( Peek et al., 2014). A limited number of inhibition/activation studies have identified dihydroxybenzoic acid and its derivatives as SDH inhibitors ( Fiedler and Schultz, 1985; Koshiba, 1978; Nandy and Ganguli, 1961), thus showing that SDH inhibitors are not limited to herbicides and organic reagents.

In this study, we chose the third strategy to identify plant SDH inhibitors among the products of the phenylpropanoid pathway (representative compounds of simple phenols, flavonoid, stilbene, and polyphenols). The strongest P. crispum SDH inhibitor was tannic acid ( Fig. 5 ). Tannins have strong astringent properties, which may induce complexation with enzymes and substrates ( Tintino et al., 2016). They bind to proteins (by hydrophobic, hydrophilic, non-specific, and specific interactions), pigments, low-molecular-weight compounds, and metallic ions (Kato et al., 2017). In microorganisms, interactions between tannic acid and the cell membrane can affect its permeability through the inhibition of the efflux pump, which may be associated with an antimicrobial effect ( Tintino et al., 2016). Furthermore, the potentially extracellular localization of tannic acid may contribute to this effect because leaf mesophyll cell walls are the typical site of origin and deposition of hydrolysable tannins in oak leaves ( Grundhofer et al., 2001). Furthermore, in the outer peels of pomegranate ( Punica granatum L.), SDHs play a role in controlling the biosynthesis of hydrolysable tannins ( Habashi et al., 2019).

Our results also showed that P. crispum SDH is inhibited at 0.15 and 0.19 mM IC 50 by caffeic acid and chlorogenic acid (with 2 and 5 hydroxyl groups in the structure), respectively. Chlorogenic acids are esters formed between caffeic acid and quinic acid, which are strong antioxidants found in many vegetable species and coffee beans ( Colon and Nerin, 2016; Guo et al., 2014; Liang and Kitts, 2015; Niggeweg et al., 2004). In plants, chlorogenic acids serve as protecting compounds against stress, e.g., viral infection ( Spoustova et al., 2015), or as feeding deterrents ( Ikonen et al., 2001). The p -coumaric, t -ferulic, sinapic, syringic, and salicylic acids, all with only one hydroxyl group, were milder SDH inhibitors, with IC 50 above 5 mM, and they are not involved in regulation under physiological conditions. On the other hand, our preliminary results indicate the presence of 0.1 μM p -coumaric, 1.7 μM t -ferulic, and 0.5 μM chlorogenic acid in P. crispum roots (data not shown) and recently Derouich et al. (2020) published a wide scale of phenolic compounds (including chlorogenic acid as the most abundant and then p -coumaric, caffeic, gallic, ferulic, vanillic, and syringic acid) in aerial parts of P. crispum plants and discussed their high antioxidant power ( Derouich et al., 2020). Considering the localization, simple phenolic compounds are probably not stored in plastids in huge amounts, modifications of cinnamic acid to p -coumaric, t -ferulic, and sinapic acid take place at the membranes of the endoplasmic reticulum, flavonoids are believed to be synthetized in the cytosol and stored in vacuoles ( Kitamura, 2006) together with monolignols derivatives, which are synthesized in the cytosol with some enzymes exhibiting membrane attachment and the bulk of the monolignol pool is targeted to the apoplast for polymerization to lignin ( Dixon and Barros, 2019). Salicylic acid is an important signal molecule; however, its concentration does not reach the value of the experimentally determined IC 50, even during stress ( Belonoznikova et al., 2020). In their study, Belinsky and Davies (1961) concluded that the both carbonyl group at the C1 position and a hydroxyl group at the 4-OH position are significant determinants of ligand binding. This is true for syringic acid with IC 50 5.1 ± 1.0 mM. Tannic acid contains several hydroxyl groups on phenyl rings; thus, their hydroxyl groups may interact with the amino acid residue in the enzyme active center.

Under non-stress conditions, plant SDH may be inhibited by some phenylpropanoid compounds. In our previous study, we found significant chlorogenic and quinic acid depletion in tobacco plants exposed to potyviral stress and heat shock ( Hyskova et al., 2021). Such a depletion could in turn favor the shikimate pathway, producing precursors of defense compounds by enhancing SDH activity.

Plant SDH inhibition by divalent metal ions, particularly Zn 2+ and Cu 2+, is known and correlated with the inactivation of functional sulfhydryl groups of SDH and also confirmed with the inhibition of plant SDH by p -chloromercuribenzoate which could be reversed by cysteine ( Balinsky and Davies, 1961; Koshiba, 1978; Lourenco and Neves, 1984). SDH from P. crispum root was also inhibited by Zn 2+ and Cu 2+, particularly by Cu 2+ ions ( Table 4 ).

Figures

Fig. 4. Identification of P. crispum SDH products by reversed-phase liquid chromatography coupled to electrospray mass spectrometry. The symbol ∅ indicates that no potential P. crispum SDH byproduct was identified in the reaction mixtures by mass spectrometry. QDH, quinate dehydrogenase; QD, quinate dehydratase; DHSD, dehydroshikimate dehydratase.

Fig. 5. Inhibition effect of various phenylpropanoids on the activity of P. crispum SDH. The specific activity of the enzyme preparations was 0.17 ± 0.07 μmol. min-1mg-1. Controls in 96 and 50 % ethanol were only slightly different from the distilled water control, with specific activities of 0.18 0.06 and 0.19 0.11 μmol. ± ± min-1mg-1, respectively. IC was calculated from nonlinear regression with Eq. (1). Each determination was done at least 3-times, the average values and standard 50 deviations are shown.