Shikimate pathway
The shikimate pathway is a seven-step metabolic route utilized by bacteria, archaea, fungi, algae, protozoans, and plants to synthesize the aromatic amino acids phenylalanine, tyrosine, and tryptophan from the central metabolites phosphoenolpyruvate and erythrose 4-phosphate.[1] This pathway converges carbohydrate metabolism with the production of essential aromatic compounds, culminating in chorismate as a key intermediate that branches into amino acid biosynthesis and secondary metabolites including ubiquinones, folates, and phenolics.[2] Absent in animals, which must obtain these amino acids from diet, the shikimate pathway's exclusivity renders it a prime target for herbicides like glyphosate, which specifically inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, disrupting aromatic compound production in target organisms.[3] Originally elucidated through isotopic labeling experiments in bacteria such as Escherichia coli during the mid-20th century, the pathway's enzymes were systematically identified, highlighting its evolutionary conservation across prokaryotes and eukaryotes excluding vertebrates.[4] Beyond amino acid provision, the shikimate pathway underpins vast metabolic diversity, serving as a precursor network for plant secondary metabolites critical to defense, signaling, and structural integrity, with implications for microbial pathogenesis and industrial biotechnology.[1]History
Discovery and Initial Characterization
The role of shikimic acid as a central intermediate in the biosynthesis of aromatic amino acids was first established in the early 1950s through experiments with bacterial auxotrophic mutants.[5] Bernard D. Davis, working at Harvard Medical School, employed penicillin enrichment techniques to isolate Escherichia coli mutants requiring phenylalanine, tyrosine, and tryptophan for growth. These mutants were observed to accumulate and excrete shikimic acid into the culture medium, indicating its position as an early precursor in the shared biosynthetic route to the three amino acids.[6] This finding built on earlier isolation of shikimic acid in 1885 from the Japanese star anise plant (Illicium anisatum), but Davis's work demonstrated its metabolic significance beyond a mere plant constituent.[7] Initial characterization of the pathway proceeded via feeding experiments with isotopically labeled precursors and analysis of accumulated compounds in blocked mutants. Davis and collaborators, including Robert L. Sprinson, identified shikimic acid as derived from carbohydrate metabolism, specifically linking phosphoenolpyruvate and erythrose-4-phosphate as initial substrates.[8] By 1955, they had outlined the core sequence involving seven enzymatic steps leading to chorismate, the branch point for aromatic amino acid synthesis, through systematic blocking of successive intermediates such as dehydroshikimate and shikimate-5-phosphate.[9] These studies, primarily in E. coli and Salmonella typhimurium, confirmed the pathway's linearity and its absence in animals, highlighting its prokaryotic and plant-specific nature.[4] Enzyme purification and assays further validated the pathway's steps during this period. For instance, the first enzyme, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, was characterized as catalyzing the condensation of phosphoenolpyruvate and erythrose-4-phosphate, with feedback inhibition by aromatic amino acids noted early on.[4] This biochemical elucidation, grounded in microbial genetics and tracer methodology, established the shikimate pathway as a distinct metabolic module bridging central carbon metabolism to aromatic compound production, setting the foundation for later genomic and structural studies.[10]Key Milestones in Elucidation
Shikimic acid, the namesake compound of the pathway, was first isolated in 1885 from the fruits of the Japanese star anise (Illicium anisatum) by Dutch chemist Johan Fredrik Eykman, though its full structure was not elucidated until the 1930s through chemical degradation and synthesis efforts.[11] [12] The biochemical role of shikimic acid remained obscure for decades, as early studies focused on its occurrence in plants rather than biosynthetic function. The pathway's elucidation began in the early 1950s through genetic and biochemical analyses of auxotrophic mutants in Escherichia coli, pioneered by Bernard D. Davis, who employed penicillin enrichment to isolate mutants blocked in aromatic amino acid synthesis.[13] These mutants accumulated shikimic acid, revealing it as a key intermediate in the de novo biosynthesis of phenylalanine, tyrosine, and tryptophan from carbohydrate precursors, distinct from mammalian pathways reliant on dietary sources.[14] Davis's 1951 publication formalized shikimic acid's central position, linking phosphoenolpyruvate and erythrose-4-phosphate as initial substrates via 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP).[14] Subsequent milestones in the 1950s and 1960s involved identifying downstream intermediates and enzymes using isotopic labeling and mutant accumulation in bacteria such as E. coli and Salmonella typhimurium. Chorismate, the pathway's branch-point intermediate, was isolated around 1956 from mutant extracts and confirmed as the precursor to all three aromatic amino acids, with its structure determined via enzymatic conversion studies. By the late 1960s, the seven enzymatic steps—from DAHP synthase to chorismate synthase—were biochemically defined, with purification of enzymes like shikimate kinase and 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase enabling mechanistic insights into phosphate transfers and dehydrations.[4] Gene cloning in the 1970s and 1980s, particularly in bacteria and yeast, allowed genetic validation of the pathway, confirming operon organization in prokaryotes and multifunctional enzymes in eukaryotes like the pentafunctional Arom complex in fungi.[4] Crystal structures of key enzymes, such as chorismate mutase in the 1990s, provided atomic-level understanding of pericyclic rearrangements, solidifying the pathway's mechanistic framework.[15] These advances, grounded in microbial genetics and enzymology, underscored the pathway's conservation across bacteria, fungi, algae, and plants, absent in animals.Biochemical Description
Pathway Steps and Intermediates
The shikimate pathway consists of seven enzymatic steps that convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), derived from glycolysis and the pentose phosphate pathway respectively, into chorismate, the branch point intermediate for aromatic amino acid biosynthesis.[1] These reactions occur primarily in the plastids of plants and the cytosol of bacteria and fungi, linking carbohydrate metabolism to the production of essential aromatic compounds.[4] The pathway's intermediates include 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS), shikimate, shikimate 3-phosphate (S3P), and 5-enolpyruvylshikimate 3-phosphate (EPSP).[16] The first committed step involves the condensation of PEP and E4P to form DAHP, catalyzed by DAHP synthase (Aro3p/Aro4p in yeast or AroF/G/H in bacteria), which is feedback-regulated by downstream aromatic amino acids.[17] Subsequent cyclization and dehydration reactions transform DAHP into DHQ via 3-dehydroquinate synthase (AroB), followed by conversion to DHS by 3-dehydroquinate dehydratase (AroD).[18] Reduction of DHS to shikimate is mediated by shikimate dehydrogenase (AroE), utilizing NADPH as a cofactor.[1] Phosphorylation of shikimate at the 3-position by shikimate kinase (AroK/L) yields S3P, which then reacts with another molecule of PEP in a reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS or AroA) to produce EPSP; this step is the target of the herbicide glyphosate, which inhibits EPSPS activity.[19] The final step involves the elimination of pyruvate from EPSP to form chorismate, driven by chorismate synthase (AroC), a pro-S-specific 1,4-elimination reaction that does not require cofactors.[17] Chorismate serves as the precursor for phenylalanine, tyrosine, and tryptophan via downstream branches.[20]| Step | Enzyme | Substrate(s) | Product | Key Features |
|---|---|---|---|---|
| 1 | DAHP synthase | PEP + E4P | DAHP | Feedback inhibition by aromatic amino acids[16] |
| 2 | 3-Dehydroquinate synthase | DAHP | DHQ | Intramolecular aldol condensation[18] |
| 3 | 3-Dehydroquinate dehydratase | DHQ | DHS | β-Elimination of water[1] |
| 4 | Shikimate dehydrogenase | DHS | Shikimate | NADPH-dependent reduction[4] |
| 5 | Shikimate kinase | Shikimate + ATP | S3P | ATP-dependent phosphorylation[17] |
| 6 | EPSPS | S3P + PEP | EPSP | Glyphosate-sensitive tetrahedral intermediate[19] |
| 7 | Chorismate synthase | EPSP | Chorismate | Cofactor-independent elimination[21] |
Enzymes and Regulation
The shikimate pathway consists of seven sequential enzymatic reactions that convert phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) into chorismate, the precursor to aromatic amino acids.[1]| Enzyme | EC Number | Reaction Catalyzed | Notes |
|---|---|---|---|
| 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) | 2.5.1.54 | PEP + E4P → DAHP + Pi | First committed step; bacterial genes aroF, aroG, aroH.[1] |
| 3-Dehydroquinate synthase (DHQS) | 4.6.1.3 | DAHP → 3-dehydroquinate (DHQ) | Multistep cyclization involving oxidation, reduction, and dehydration.[1] |
| 3-Dehydroquinate dehydratase (DHQD) | 4.2.1.10 | DHQ → 3-dehydroshikimate (DHS) | Type I (Schiff base mechanism) or Type II (zinc-dependent); bacterial gene aroD.[1] |
| Shikimate dehydrogenase (SDH) | 1.1.1.25 | DHS + NADPH → shikimate + NADP⁺ | In plants, often bifunctional with DHQD.[1] |
| Shikimate kinase (SK) | 2.7.1.71 | Shikimate + ATP → shikimate-3-phosphate (S3P) + ADP | Bacterial gene aroL; ATP-dependent phosphorylation.[1] |
| 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) | 2.5.1.19 | S3P + PEP → EPSP + Pi | Target of herbicide glyphosate; bacterial gene aroA.[1] |
| Chorismate synthase (CS) | 4.2.3.5 | EPSP → chorismate | Eliminative rearrangement; bacterial gene aroC.[1] |