Structural and Biochemical Analyses Reveal that Chlorogenic Acid Inhibits the Shikimate Pathway

Abstract

<jats:p>Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization display the inhibitory role of CGA against the enzyme of the shikimate pathway, a well characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from <jats:italic>Providencia alcalifaciens</jats:italic> (<jats:italic>Pa</jats:italic>DHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of <jats:italic>Pa</jats:italic>DHQS, which disables domain movements, leaving the enzyme in open and catalysis incompetent state. The binding analyses by ITC and SPR show that CGA binds to <jats:italic>Pa</jats:italic>DHQS with a <jats:italic>K</jats:italic><jats:sub>D</jats:sub> value of 6.3 μM and 0.5 μM respectively. <jats:italic>In vitro e</jats:italic>nzyme inhibition studies show that CGA inhibits <jats:italic>Pa</jats:italic>DHQS with a <jats:italic>K</jats:italic><jats:sub>i</jats:sub> of 235 ± 21 μM; while it inhibits the growth of <jats:italic>Providencia alcalifaciens</jats:italic>, <jats:italic>Moraxella catarrhalis</jats:italic>, <jats:italic>Staphylococcus</jats:italic> <jats:italic>aureus</jats:italic> and <jats:italic>Escherichia coli</jats:italic> with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA doesn't show the toxic effect. These results, along with the observations of the inhibition of DAHP regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis, and can be further exploited for designing novel drug-like molecules.</jats:p> <jats:p><jats:bold>Importance</jats:bold> The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents as it is essential in plants, bacteria and apicomplexan parasites, but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway will act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of shikimate pathway. The crystal structure of dehydroquinate synthase the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explain the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for the further development as potent inhibitor of shikimate pathway enzymes.</jats:p>

Scrutinizing the Antimicrobial and Antioxidant Potency of European Cranberry Bush (Viburnum opulus L.) Extracts

“…However, it has been proposed that the loss of cell components only partially affects the viability of microorganisms. Some intracellular processes in bacteria, apart from cell wall permeabilization, lie behind the antimicrobial activity of chlorogenic acid [87]. The observations revealed the ability of chlorogenic acid to bind to shikimate pathway enzymes with high affinity and to inhibit their catalysis, thus causing irreversible cell death.…”

Molecular mechanisms underlying glyphosate resistance in bacteria

“…In addition to glyphosate, other substances have been identified and characterized in recent years that selectively inhibit the shikimate pathway (Fig. 1A; Han et al ., 2006; Zhu et al ., 2018; Brilisauer et al ., 2019; Neetu et al ., 2020). A very promising substance is the deoxy sugar 7‐deoxy‐sedoheptulose (7dSH) that is produced by the cyanobacterium Synechococcus elongatus and inhibits the dehydroquinate synthase (Fig.…”

“…The shikimate pathway and substances inhibiting its enzymes. 7‐Deoxy‐seduheptulose (7dSH) and chlorogenic acid were shown to inhibit the dehydroquinate synthase (Brilisauer et al ., 2019; Neetu et al ., 2020). IMB‐T130, curcumin and glyphosate inhibit the dehydroquinate dehydratase (Zhu et al ., 2018), the shikimate dehydrogenase (Han et al ., 2006) and the enolpyruvyl 3‐phosphate (EPSP) synthase (Amrhein et al ., 1980) respectively.…”

Molecular analysis and essentiality of Aro1 shikimate biosynthesis multi-enzyme in Candida albicans

“…In plants and bacteria these activities are typically performed by individual single-domain enzymes: 3-dehydroquinate synthase (DHQS), 3-dehydroquinate dehydratase type I (DHQase), 3-dehydroshikimate dehydrogenase (DHSD), shikimate kinase (SK), and 5-enolpyruvate-shikimate-3-phosphate synthase (EPSPS). DHQS (Liu et al, 2008;Neetu et al, 2020) and type I DHQase (Nichols et al, 2004b;Light et al, 2011).…”