A HR-MS Based Method for the Determination of Chorismate Synthase Activity

Khera, Harvinder Kour; Singh, S. K.; Mir, Riyaz Ahmad; Bharadwaj, Vikram; Singh, Subhash

doi:10.2174/0929866523666161222153707

Cited by 6 publications

(2 citation statements)

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“…In the present study, we analysed the localization of PhCS and obtained a PhCS-silenced petunia phenotype using VIGS, and our results demonstrated that CS plays important roles in plant growth and development. Full-length cDNAs for CSs have been isolated from a number of organisms, including Arabidopsis, Corydalis sempervirens, tomato, Euglena gracilis, and Mycobacterium tuberculosis 27,[56][57][58] . Studies have indicated that CSs are encoded by a single gene or a small gene family 28 .…”

Section: Discussionmentioning

confidence: 99%

Suppression of chorismate synthase, which is localized in chloroplasts and peroxisomes, results in abnormal flower development and anthocyanin reduction in petunia

Zhong

Chen

Han

et al. 2020

Sci Rep

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in plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. chorismate synthase (cS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a singlecopy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. petunia plants with reduced phcS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia. The shikimate pathway provides the basic building blocks for the synthesis of three aromatic amino acids as well as an array of aromatic secondary metabolites, such as flavonoids, alkaloids, and lignins. This pathway is highly conserved in fungi, bacteria, and plant species 1-3 and consists of seven metabolic steps, and six key enzymes, including 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS), 3-dehydroquinate synthase (DHQS), 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH), shikimate kinase (SK), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, and chorismate synthase (CS), are involved in these steps (Fig. 1). Previous studies have indicated that many enzymes involved in the shikimate pathway have been found in plastids 2,4-12. In addition, most shikimate pathway enzymes are synthesized as precursors with an N-terminal extension, which allows the post-translational uptake of the protein into chloroplasts 13,14. In addition, the use of green fluorescent protein (GFP) fusion proteins or protein import assays has confirmed the plastidic localization of most enzymes in this pathway, such as DAHPS, anthranilate synthase, EPSPS, DHD/SDH, and SK, with the exception of CS 13-22. However, not all enzymes involved in the shikimate pathway are located in plastids. The isoforms of some enzymes involved in the shikimate pathway, such as DAHPS 23 and EPSPS 4 , lack N-terminal transit peptides 24 .

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Section: Discussionmentioning

confidence: 99%

Suppression of chorismate synthase, which is localized in chloroplasts and peroxisomes, results in abnormal flower development and anthocyanin reduction in petunia

Zhong

Chen

Han

et al. 2020

Sci Rep

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“…A new discontinuous method for measuring CS activity has been proposed to be applicable to both monofunctional (anaerobic) and bifunctional (aerobic) enzymes [153]. In short, the method is comprised of in situ production of EPSP by recombinant MtEPSPS, ultrafiltration, addition of FMN, NADH and recombinant MtCS, filtration and detection of chorismate by liquid chromatography coupled with negative electrospray ionization high-resolution tandem mass spectrometry (ESI-HRMS).…”

Section: Chorismate Synthase (Arof Coding Sequence; Ec 4235)mentioning

confidence: 99%

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

et al. 2020

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Roughly a third of the world’s population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.

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