Shikimate biosynthesis

Shikimic acid or shikimate is an important metabolite in plants and micro organisms and absent in animals. Shikimate is important for many biosynthetic processes including that of folate, aromatic amino acids and ubiquinone. This is because chorismate, the precursor for these pathways is synthesised from shikimate. Chorismate is de novo synthesised from erythrose-4-phosphate, a product of pentose phosphate cycle and phosphoenolpyruvate, a product of glycolysis via the intermediate shikimate in seven reactions. The steps 2-6 of shikimate biosynthesis pathway (4.2.3.4, 4.2.1.10, 1.1.1.25, 2.7.1.71 & 2.5.1.19 respectively) is carried out by a penta-functional protein called AROM peptide in yeast [1] and all these activities are annotated to a single protein (TGME49_107040/NCLIV_053120) in Toxoplasma  gondii and Neospora caninum. The InterProScan shows the presence of functional domains for all these activities in this protein. The Plasmodium falciparum gene models show that the first three enzymes of AROM peptide  (steps 2-4) are missing, whereas the bifunctional protein PFB0280w possesses last two activities. It has been suggested back in 2002 by Gardner et al that the there has been a predicted protein with low sequence similarity to AROM peptide observed [2]. Although these enzymes are missing in the genome, Shikimate biosynthesis pathway is reconstructed in MPMP on the basis of experimental evidences available.

 

The first of these evidences came from P. falciparum auxotrophic strains which depends on exogenous supply of 4-aminobenzoate (PABA) in contrast to wild type strains [3]. The treatment with shikimate analogues, (6R)-6-Fluoro-shikimate and (6S)-6-fluoro-shikimate has resulted in inhibition of P. falciparum growth and reversal of it with PABA suggested the specific targeting of shikimate pathway [4].The presence of shikimate pathway was also demonstrated in T. gondii and P. falciparum by treating the parasites with the herbicide glyphosate, an inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase enzyme. This has resulted in the inhibition of growth of in vitro T. gondii tachyzoites and erythrocyte stages of P. falciparum. This effect was reversible with addition of PABA or folate in medium suggesting the role of shikimate pathway in providing precursors for folate biosynthesis. The glyphosphate inhibition of growth was also observed in Cryptosporidium parvum and the effect was not recovered with folate or PABA suggesting that this pathway does more than simply providing folate precursors in C. parvum [5]. In addition, the enzymes chorimsate dehydrogenase, 3-dehydroquinate dehydratase and shikimate kinase was also detected in T. gondii extracts and the enzyme chorimsate synthase was cloned from both  P. falciparum and T. gondii. Sulfonamide, a component of anti-malarial drug Fansidar competes with PABA for dihydropteroate synthase and the dihydropteroate anologue synthesised from sulfonamide  is toxic and inhibits dihydrofolate synthase enzyme. The comparison of sulfonamide-resistant and sensitive strains of P. falciparum demonstrated that the resistant strain does not require PABA from medium, whereas sensitive strains uptake PABA. This also confirms the presence of shikimate de novo biosynthesis pathway and its role in providing PABA for folate biosynthesis. In addition, four enzymes of shikimate pathway namely 3-deoxy-7-phosphoheptulonate synthase, shikimate dehydrogenase, shikimate kinase and aminodeoxy-chorismate lyase (PABA synthetase) were detected in both sensitive and resistant strains of P. falciparum. Of these, the former three enzymes were found in similar amounts, whereas only PABA synthetase was detected 2.5 fold higher in resistant strains confirming that it can compensate by upregulating synthesis of PABA from chorismate [6]. These results suggest that complete shikimate pathway is present and it can be an effective drug target for Malaria, Toxoplasmosis, Neosporosis and Cryptosporidiosis. Although these results confirm the importance of this pathway in providing PABA for folate biosynthesis, the role of shikimate pathway in ubiquinone biosynthesis is not clear.

 

Enzyme EC Number Gene id
3-deoxy-7-phosphoheptulonate synthase 2.5.1.54 NCLIV_004821
Aminodeoxy-chorismate synthase/Aminodeoxy-chorismate lyase/Anthranilate synthase 2.6.1.85; 4.1.3.27 NCLIV_022000
Aminodeoxy-chorismate lyase 4.1.3.38 Missing in annotation
Pentafunctional 3-dehydroquinate synthase/3-dehydroquinate dehydratase/shikimate dehydrogenase/shikimate kinase/5-Enolpyruvylshikimate-3-phosphate synthase 4.2.3.4; 4.2.1.10; 1.1.1.25; 2.7.1.71; 2.5.1.19 NCLIV_053120
Chorismate synthase 4.2.3.5 NCLIV_023120

 

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Sources and fates of metabolites

 

Substrate Source pathways Product Fate pathways
D-Erythrose-4-P Pentose phosphate cycle Chorismate Ubiquinone metabolism
Phosphoenolpyruvate Glycolysis 4-Aminobenzoate Folate biosynthesis
    Anthranilate ?