Shikimate dehydrogenase (EC 1.1.1.25), encoded by the aroE gene in Escherichia coli, catalyzes the reversible reduction of 3-dehydroshikimate to shikimate, using NADPH as a cofactor. This is the fifth step in the shikimate pathway, critical for microbial viability and absent in mammals—making AroE a target for antimicrobial drug development.

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The term dehydratase-synthase refers to enzymes that perform dehydration and condensation reactions, often in biosynthetic pathways such as polyketide synthesis, fatty acid metabolism, or amino acid biosynthesis. In the shikimate pathway, such enzymatic activities assist in ring closures and intermediate transformations.

1. Metabolic Pathway Reconstruction

Recombinant AroE and dehydratase enzymes are used to reconstruct the shikimate pathway in heterologous hosts like E. coli, yeast, and Corynebacterium glutamicum to produce:

• Aromatic amino acids
• Folate intermediates
• Antibiotics (e.g., chloramphenicol)
• Anticancer agents (e.g., shikimic acid derivatives)

2. Antimicrobial Dr ug Target Screening

Because the shikimate pathway is absent in humans, AroE is studied as a selective dru g target for:

• Mycobacterium tuberculosis
• Pseudomonas aeruginosa
• Staphylococcus aureus

Recombinant AroE is typically cloned into a plasmid vector (e.g., pET, pBAD), expressed in E. coli BL21 (DE3), and purified using :

• His-tag affinity chromatography
• Ni-NTA resin columns
• Size-exclusion chromatography (SEC)

Protein purity is confirmed using SDS-PAGE and Western blotting, and activity is measured via NADPH consumption assays at 340 nm.