Lecture 8 Shikimate Pathway Dr. Normah 2024 PDF

Shikimate pathway: aromatic amino acids and phenylpropanoids (02/5/2024) Assoc. Prof. Ts. Dr. Normah binti Haron The shikimate pathway Defined as 7 metabolic steps C3H5O6P C4H9O7P 6 enzymes C7H13O10P C10H10O6 Pathway intermediates and e...

Shikimate pathway: aromatic amino acids and phenylpropanoids (02/5/2024) Assoc. Prof. Ts. Dr. Normah binti Haron The shikimate pathway Defined as 7 metabolic steps C3H5O6P C4H9O7P 6 enzymes C7H13O10P C10H10O6 Pathway intermediates and enzymes of shikimate pathway  First enzyme of the shikimate pathway. It catalyzes the condensation of phosphoenolpyruvate (PEP) and erythrose4-phosphate (Ery4P) to yield DAHP. i. 3-deoxy-o- The most intensively investigated DAHP synthase is arabino- the enzyme from E. coli. heptulosonate- Wild type E. coli produces three feedback inhibitor- 7-phosphate sensitive DAHP synthase isoenzymes: a Tyr-sensitive, a Phe-sensitive and a Trp-sensitive enzyme. synthase This multi-isoenzyme systemàensures a sufficient supply of chorismate for the biosynthesis of other aromatic compounds when Tyr, Phe and Trp are present in excess in the growth medium.  It catalyzes the elimination of phosphate from DAHP to generate 3-dehydroquinate (DHQ). The enzyme from E. coli requires divalent cations for activity. ii. 3- DHQ synthase from E. coli is activated by inorganic dehydroquinate phosphate, and it also requires catalytic amounts of NAD synthase for activity, although the enzyme catalyses a redox neutral reaction (Bender et al., 1989). Unlike DAHPS, potato DHQ synthase activity does not change when cells are exposed to glyphosate (Pinto et al., 1988). iii. 3-dehydroquinate dehydratase- shikimate dehydrogenase The third step of the shikimate pathway, the dehydration of DHQ to form 3- dehydroshikimate (DHS), is catalysed by DHQ dehydrogenase (DHD). There are two types of DHD in bacteria. The type I enzyme-heat labile, and has Km values in the lower micromolar range. The type II enzyme-heat stable, and the Km of the type II enzyme is one or two orders of magnitude higher as compared to the type I enzyme. There is no sequence similarity between these two types of isoenzymes. Most bacteria and plants only have the type I enzyme of DHD, whereas fungi have both. Continue… In general, the type I enzyme participates in the anabolic reaction of the shikimate pathway, and the type II enzyme participates in the catabolic reaction of the quinate pathway (Giles et al., 1985). iv. Shikimate kinase E. coli has two isoenzymes of shikimate kinase, and both can function in vitro for aromatic amino acid biosynthesis. Isoenzyme II has a Km of 200 µM for shikimate, Isoenzyme I is more than 100 times higher than isozyme II v Thus, in vivo, isoenzyme I might not be an enzyme of the shikimate pathway. Plant shikimate kinase has been described for spinach and tomato. The shikimate kinase gene is expressed in an organ-specific manner similar to the expression of EPSP synthase and chorismate synthase. The abundance of shikimate kinase transcripts was highest in flowers, lower in roots and even lower in stems, cotyledons and leaves v. 5-enolpyruvyl-shikimate-3-phosphate synthase It catalyzes the reversible formation of 5-enolpyruvyl-shikimate-3- phosphate (EPSP) from shikimate 3-phosphate and PEP. Plant cDNAs encoding EPSPS have been isolated from petunia, Arabidopsis, tomato, and tobacco. All cDNAs encode precursor proteins with N-terminal transit sequences for plastid import. In vivo uptake experiments have shown that the petunia transit sequence directs proteins into plastids. This enzyme has been deeply analysed because it is the target of a broad-spectrum herbicide, glyphosate (N- [phosphonomethyl]-glycine). When challenged with glyphosate, plant cells would accumulate or excrete shikimate High level accumulation of shikimate in the glyphosate-treated plants may be the result of a deficiency of feedback control of the shikimate pathway vi. Chorismate synthase It catalyzes the trans-1, 4 elimination of phosphate from EPSPS to yield chorismate. In this reaction, the second of three double bonds of the benzene ring is introduced. Chorismate synthase requires a reduced flavin nucleotide (FMNH2) as a cofactor, even though the overall reaction is redox neutral. In this aspect, the enzyme is similar to DHQ synthase, the second enzyme in the shikimate pathway. Like other shikimate pathway enzymes, the cDNA of chorismate synthase encodes a precursor protein with N-terminal transit sequences for plastidic targeting. Chorismate synthase activity requires cleavage of the transit peptide. In contrast, other shikimate pathway enzymes (shikimate kinase, DHD/SHD and EPSP synthase) have been shown to be active in both immature and mature forms.  The demand for phenolic metabolites varies during plant development and during periods of environmental stress such as wounding or pathogen attack. Regulation of the Therefore, mechanisms that control carbon flow into the shikimate pathway shikimate must perceive and respond accordingly. pathway For both plants and bacteria, at least two of the shikimate pathway enzymes, DAHP synthase and shikimate kinase can be regulated.  The prokaryotic DAHP synthase is regulated by feedback inhibition. The genes encoding the Tyr- and Trp-sensitive isoenzymes are regulated by Tyr and Trp, respectively , whereas the Trp-sensitive isoenzyme is regulated by both amino acids In contrast to bacterial DAHPS, plant DAHP synthases are not subject to feedback inhibition by the aromatic amino acids.  In plants, proteins are synthesized in 3 different compartments: in the cytoplasm, in the plastids Subcellular and in the mitochondria. localization The aromatic amino acids must either be of the synthesized in situ in the respective protein- synthesizing compartments or synthesized outside shikimate the compartments and have to be imported. pathway The isolated chloroplasts from spinach were able to synthesize aromatic amino acids from CO2 or shikimateàindicated the existence of an aromatic biosynthetic pathway in chloroplasts.  The localization of the shikimate pathway in the plastids is supported by molecular analysis. In addition, in vitro experiments demonstrated that some shikimate pathway enzymes (e.g. shikimate kinase and EPSPS) can be imported into chloroplasts Thus, the shikimate pathway appears to be firmly established in plastids. However, it has been proposed that the chloroplast localized biosynthetic activity does not count for 100% of the observed aromatic amino acid biosynthesis. Assignment- Dr. Normah’s part 10 marks You’ll be given 3 papers. A short quiz will be conducted virtually based on the papers. Proposed date is on Week 13 (during class time).